An Efficient Beet Severe Curly Top Virus‐Based Vector for VIGS and HIGS in Beta vulgaris

Sugar beet (Beta vulgaris L.), one of the most important sugar crops worldwide, contributes approximately 25% of global sugar production. However, genomic studies in sugar beet have been hindered by the lack of efficient genetic tools. Virus-induced gene silencing (VIGS), a widely used technique for genomic studies in plants (Liu et al. 2002), has shown applicability in inoculated leaves of sugar beet (Hatlestad et al. 2015). Nonetheless, the development of a highly efficient VIGS system remains an urgent need. Beet severe curly top virus (BSCTV) is a monopartite virus belonging to the genus Curtovirus (family Geminiviridae). BSCTV has a very broad host range within 44 dicot plant families (Strausbaugh et al. 2008). Thus, BSCTV is a promising candidate for engineering VIGS vectors for functional genomic studies in sugar beet and other plant species. In this study, we first constructed an infectious cDNA clone pCB-BSCTV (Figure 1a) by inserting 1.2 copies of the BSCTV genome (GenBank accession: U02311) into a binary vector pCB301. All primers used in this study were listed in Table S2. Agrobacterium tumefaciens containing pCB-BSCTV was infiltrated into leaves of the sugar beet variety BETA176. At 12 days post-inoculation (dpi), the rescued BSCTV induced severe leaf curling symptoms (Figure 1b) and exhibited high infectivity in the systemic leaves and roots of sugar beet (Figure S1a). Previous studies have identified that BSCTV C2 protein is a determinator of viral pathogenicity and the C1/C2/C3 overlapping region generates abundant of viral small interfering RNAs (vsiRNAs) (Zhang et al. 2011; Voorburg et al. 2021). Therefore, we selected the overlapping region as the insertion site for the VIGS vector, anticipating that it would attenuate viral symptoms and enhance RNA silencing efficiency. To develop an effective VIGS system, we inserted 300 bp fragments of either GFP or B. vulgaris Phytoene desaturase (BvPDS, XM_010686886) into the C1/C3 junction of pCB-BSCTV (Figure 1a). As expected, both BSCTV-GFP and BSCTV-BvPDS induced attenuated symptoms due to lack of the C2 coding frame (Figure 1c). Notably, systemically infected leaves of sugar beet inoculated with BSCTV-BvPDS exhibited bleaching phenotype at 20 dpi (Figure 1c). Subsequently, the bleached phenotype was extended to all newly emerging leaves (Figure 1d). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results revealed a significantly reduced BvPDS transcript levels in BSCTV-BvPDS-infected plants compared to BSCTV-GFP controls (Figure 1e). Both inserted fragments of GFP and BvPDS remained genetically stable at 30 dpi (Figure 1f). In four independent experiments, approximately 82% of inoculated sugar beet plants (n = 55) exhibited bleached phenotype (Table S1). We also confirmed that 180 bp BvPDS fragment insertion was sufficient to induce effective gene silencing (Figure S1b). The BSCTV-BvPDS system showed broad applicability across eight tested sugar beet varieties, with particularly strong silencing observed in SV1434 and table beet, which exhibited complete photobleaching (Figure 1g). These results collectively establish BSCTV-VIGS as an effective tool for genomic studies in sugar beet. To further validate the versatility of the BSCTV-based VIGS system, we targeted B. vulgaris MYB1 (BvMYB1, JF432079), a key transcriptional regulator of the betalain pathway in sugar beet (Hatlestad et al. 2015). To this end, a 300 bp BvMYB1 fragment was inserted into pCB-BSCTV and delivered into sugar beet plants via agroinfiltration. At 35 dpi, plants infected with BSCTV-BvMYB1 displayed green veins and stems of systemic leaves compared with the BSCTV-GFP control (Figure 1h). RT-qPCR results confirmed a significant down-regulation of BvMYB1 transcription levels in leaf tissues compared to those of BSCTV-GFP infected plants (Figure 1i). These findings demonstrate that the BSCTV-based VIGS system can be applied to gene function studies in leaf and stem of sugar beet plants. To explore the potential of the BSCTV system for host-induced gene silencing (HIGS), we targeted vital genes of Cercospora beticola, the causal agent of the most devastating foliar disease in sugar beet. We engineered the recombinant BSCTV vector to silence CbNip1 (XM_023596625) of C. beticola, a key fungal effector gene (Ebert et al. 2021). Seedlings of BETA176 were infiltrated with BSCTV-CbNip1 or BSCTV-GFP. At 40 dpi, we inoculated upper leaves with C. beticola by spraying method (Ebert et al. 2021). Remarkably, BSCTV-CbNip1-infected plants exhibited dramatically attenuated disease symptoms compared to controls at 10 dpi (Figure 1j). In consistence, qPCR results showed significantly reduced fungal biomass in BSCTV-CbNip1 infected plants compared to the BSCTV-GFP control (Figure 1k). Moreover, substantially down-regulated CbNip1 transcript levels (Figure 1l), confirming the efficient cross-kingdom RNA silencing. These results demonstrate that the BSCTV-VIGS system can deliver functional small RNAs across kingdom boundaries to silence critical fungal genes for dissecting sugar beet-pathogen interactions. In summary, we have developed an efficient and user-friendly BSCTV-based VIGS and HIGS systems for sugar beet, representing a significant advancement in functional genomics tools for this important crop. In addition, this versatile platform will greatly facilitate VIGS development in other economically important crops. Future research directions will focus on adapting the BSCTV vector into a virus-induced genome editing (VIGE) system, analogous to the recently developed barley yellow striate mosaic virus (BYSMV)-based ViGET platform in wheat (Qiao et al. 2025). Y.W., C.G.-H., Z.Z. and Z.N. designed the experiments. Z.N., Z.G., X.Q., Q.L. and M.Z. conducted the experiments and analysed the data. C.G.-H. and J.Y. contributed to the discussion of the data. Z.N., X.-B.W. and Y.W. wrote the manuscript. All authors discussed the results and commented on the manuscript. We thank Professors Jialin Yu, Dawei Li and Yongliang Zhang (China Agricultural University, Beijing, China) for their helpful comments on the research. We also thank Professor Xiaorong Tao (Nanjing Agricultural University, Nanjing, China) for providing the binary vector pCB301-2x35S-MCS-HDVRZ-NOS, Professor Huizhong Zhang (Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot, China) for providing sugar beet varieties. This work was supported by the National Natural Science Foundation of China (32270165 and 31872921) and China Agricultural Industry Technology System (Grant No. CARS-170304). The authors have nothing to report. Table S1: The silencing efficiency of BSCTV-BvPDS in sugar beet BETA176. Table S2: The primers used in this study. Data S1: pbi70345-sup-0003-Supinfo.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.