Pepper is an important vegetable cultivated worldwide. Pepper fruits accumulate unique metabolites, capsanthin and capsaicin, which are important raw materials for natural pigment and medicine. Pepper plants are rich in genetic diversity and are attractive subjects for fruit developmental studies. The release of pepper reference genomes provided key genetic information for dissecting function of pepper genes underlying various interesting phenotypes (Kim et al., 2014; Qin et al., 2014). However, due to the difficulty in transformation, dissecting genetic mechanisms in pepper remained technically challenging. RNA silencing is a small RNA-mediated gene silencing mechanism operating in most eukaryotes and working as a natural antiviral defence mechanism (Ding, 2010). Virus-induced gene silencing (VIGS) not only targets invading virus, but also can repress expression of plant genes, and thus, many viruses were developed into VIGS tools for studying plant gene function. Tobacco rattle virus (TRV) was a well-developed VIGS vector and widely used in plants (Ratcliff and Martin-Hernandez, 2001; Shi et al., 2021). TRV-based VIGS was frequently used in pepper, but the efficiency was not optimized, particularly for silencing gene expression in flowers and fruits.

In our initial effort to optimize VIGS efficiency in pepper leaves, we made the pTRV2-GFP-CaPDS vector from the pTRV2-LIC vector (Dong et al., 2007), which allowed us visualize TRV spreading and monitor VIGS efficiency (Figure 1a), and determined optimal experimental condition, in which agrobacterium of OD₆₀₀ 0.004 was infiltrated on cotyledon of two-week-old seedlings and resulted 60% of treated plants showing photobleaching. Successful VIGS depends on efficient virus movement in plants, which is controlled impart by antiviral RNA silencing and viral suppressors (VSR) (Li and Ding, 2006). We reasoned that expression of heterologous VSRs from TRV may enhance its spreading in pepper, thus enhancing VIGS efficiency. To test this hypothesis, a panel of pTRV2 vectors were made from pTRV2-GFP-CaPDS with GFP in-frame fused to various VSRs, including citrus tristeza virus CP, P23 and P20, tomato bush stunt virus P19, cucumber mosaic virus 2b (C2b) and tomato aspermy virus 2b (T2b) (Li and Ding, 2006) (Figure 1a). These vectors were co-expressed with pTRV1 in pepper cotyledon by agroinfiltration. Green florescence imaging and Western blot analysis showed that all TRV viruses replicated and spread well in pepper plants (Figure 1b,c). Statistics analysis showed that C2b-expressing vector caused PDS silencing in 93% of treated plants, resulting in highest VIGS efficiency among all vectors (Figure 1b). With the high efficiency TRV2-GFPC2b-CaPDS vector, we screened our pepper germplasm and identified about 30 accessions with PDS silencing efficiency ranging from 70% to 100%. C2b (from subgroup II CMV-Q, X00985) showed the best performance in promoting VIGS efficiency, which is consistent with its three merits: (i) it has strong systemic silencing suppressor activity that promotes virus spreading; (ii) it has a weak local silencing suppressor activity that will not affect silencing of target genes in target cells; (iii) it has short coding sequences that minimizes fitness cost brought to TRV vector (Guo and Ding, 2002). These merits made C2b an idea VSR to promote TRV spreading in pepper and induce target gene silencing in systemically infected leaves.

To test how the optimized TRV vector performs in pepper flowers, pTRV2-C2b-CaAGL vector was made to target pepper AGAMOS (AG) gene in which GFP ORF was removed to increase virus fitness (Figure 1a). Four pepper accessions with good PDS-VIGS efficiency were infected with pTRV1 and pTRV2-C2b-CaAGL. About 6 weeks after infiltration, altered flower phenotype appeared among all the four pepper accessions, while L085 and L265 accessions showed better overall performance with good flower setting and silencing efficiency (81% and 84% respectively). Persistence of TRV infection in pepper flowers and knocking-down of CaAG expression were confirmed by RT-PCR (Figure 1d,e). Close examination showed different types of flower structure on pTRV2-C2b-CaAGL infected pepper, which were different from reported Arabidopsis ag null mutant and RNAi-lines (Figure 1f) (Bowman and Smyth, 1989; Mizukami and Ma, 1995). VIGS of AG in Arabidopsis also resulted in abnormal floral structures that are different from its CRISPR mutant (Figure 1f). Although VIGS of AG revealed its dual roles in petal identity and floral determinacy in both species, the floral determinacy in pepper and Arabidopsis appeared different (Figure 1f). These results suggested that our optimized VIGS method can silence genes efficiently in pepper flowers and also revealed unique advantage for VIGS in studying developmental genes.

To test VIGS efficiency in pepper fruit, pTRV2-C2b-CaCCS vector was constructed (Figure 1a), which targets the key gene in capsanthin/capsorubin biosynthesis (Kim et al., 2014). Five red pepper accessions (L085, L099, L228, L252 and L265), suitable for long-term maintenance in growth room, were infected with pTRV1 and pTRV2-C2b-CaCCS by agroinfiltration. The TRV-infected plants were regularly watered and fertilized till fruit maturation, which took 14–16 weeks after infiltration. The results showed that 97% and 100% of pTRV2-C2b-CaCCS infected L085 and L228 plants produced yellow fruits while no plants infected with pTRV2-C2b produced yellow fruits, indicating high efficiency of CaCCS silencing in fruit (Figure 1g). Knocking-down of CaCCS transcript levels and persistence of TRV infection in the yellow fruit were confirmed by RT-PCR (Figure 1d,h). These results suggested that the optimized TRV vector and selected pepper accessions could constitute an efficient VIGS system for studying gene function in pepper fruits.

In summary, our study optimized TRV-based VIGS methods in pepper and provided highly efficient vectors and pepper accessions that could facilitate studies of gene function throughout pepper life cycle. And the strategy could also be applied to other plant VIGS system.

胡椒是世界范围内种植的重要蔬菜。辣椒果实中积累了独特的代谢产物辣椒红素和辣椒素，是天然色素和药物的重要原料。辣椒植物具有丰富的遗传多样性，是果实发育研究的有吸引力的课题。辣椒参考基因组的发布为剖析各种有趣表型背后的辣椒基因功能提供了关键的遗传信息（Kim et al ., 2014 ; Qin et al ., 2014）。然而，由于转化困难，剖析辣椒的遗传机制在技术上仍然具有挑战性。RNA 沉默是一种小 RNA 介导的基因沉默机制，在大多数真核生物中发挥作用，并作为一种天然的抗病毒防御机制 (Ding, 2010 )。病毒诱导的基因沉默（VIGS）不仅针对入侵病毒，还可以抑制植物基因的表达，因此许多病毒被开发成研究植物基因功能的VIGS工具。烟草响尾病毒 (TRV) 是一种成熟的 VIGS 载体，广泛用于植物中 (Ratcliff 和 Martin-Hernandez, 2001 ; Shi et al ., 2021）。基于 TRV 的 VIGS 经常在辣椒中使用，但效率并未优化，特别是对于沉默花和果实中的基因表达。

在我们优化辣椒叶中 VIGS 效率的最初努力中，我们从 pTRV2-LIC 载体（Dong等人，2007 年）制作了 pTRV2-GFP-CaPDS 载体，这使我们能够可视化 TRV 传播并监控 VIGS 效率（图 1a） , 并确定了最佳实验条件，其中 OD ₆₀₀ 0.004 的农杆菌渗透到两周龄幼苗的子叶上，导致 60% 的处理植物出现光漂白。成功的 VIGS 依赖于植物中有效的病毒运动，这是由抗病毒 RNA 沉默和病毒抑制因子 (VSR) 控制的 (Li and Ding, 2006）。我们推断，来自 TRV 的异源 VSR 的表达可能会增强其在辣椒中的传播，从而提高 VIGS 效率。为了验证这一假设，一组 pTRV2 载体由 pTRV2-GFP-CaPDS 制成，其中 GFP 框内融合到各种 VSR，包括柑橘树病毒 CP、P23 和 P20、番茄灌木矮化病毒 P19、黄瓜花叶病毒 2b (C2b ) 和番茄无精子症病毒 2b (T2b) (Li and Ding, 2006) (图 1a)。这些载体通过农杆菌浸润在辣椒子叶中与 pTRV1 共表达。绿色荧光成像和蛋白质印迹分析表明，所有 TRV 病毒在辣椒植物中复制和传播良好（图 1b，c）。统计分析表明，表达 C2b 的载体在 93% 的处理植物中引起 PDS 沉默，导致所有载体中 VIGS 效率最高（图 1b）。利用高效的TRV2-GFPC2b-CaPDS载体，我们筛选了辣椒种质，鉴定了大约30个PDS沉默效率在70%到100%之间的种质。C2b（来自亚组 II CMV-Q，X00985）在提高 VIGS 效率方面表现出最佳性能，这与其三个优点一致：（i）它具有促进病毒传播的强大的全身性沉默抑制活性；(ii) 它具有较弱的局部沉默抑制活性，不会影响靶细胞中靶基因的沉默；(iii) 它的编码序列很短，可以最大限度地降低 TRV 向量的适应度成本（Guo 和 Ding，2002 年）。这些优点使 C2b 成为一种想法 VSR，以促进 TRV 在辣椒中传播并在全身感染的叶子中诱导靶基因沉默。

为了测试优化的 TRV 载体在辣椒花中的表现，pTRV2-C2b-CaAGL 载体被制成靶向辣椒AGAMOS ( AG ) 基因，其中去除了 GFP ORF 以增加病毒适应度（图 1a）。用 pTRV1 和 pTRV2-C2b-CaAGL 感染了四个具有良好 PDS-VIGS 效率的辣椒种质。浸润后约 6 周，所有 4 个辣椒种质都出现了改变的花表型，而 L085 和 L265 种质表现出更好的整体性能，具有良好的开花和沉默效率（分别为 81% 和 84%）。辣椒花中TRV感染的持续性和CaAG的敲除通过 RT-PCR 确认表达（图 1d，e）。仔细检查显示 pTRV2-C2b-CaAGL 感染辣椒上不同类型的花结构，这与报道的拟南芥 ag无效突变体和RNAi系不同（图 1f）（Bowman 和 Smyth，1989；Mizukami 和 Ma，1995）。拟南芥中AG的VIGS也导致与其 CRISPR 突变体不同的异常花结构（图 1f）。虽然AG的 VIGS揭示了它在两个物种的花瓣特性和花决定性中的双重作用，辣椒和拟南芥的花决定性似乎不同（图1f）。这些结果表明，我们优化的 VIGS 方法可以有效地沉默辣椒花中的基因，也揭示了 VIGS 在研究发育基因方面的独特优势。

为了测试辣椒果实中的 VIGS 效率，构建了 pTRV2-C2b-CaCCS 载体（图 1a），该载体靶向辣椒红素/辣椒红素生物合成中的关键基因（Kim et al ., 2014）。采用农杆菌浸润法感染 pTRV1 和 pTRV2-C2b-CaCCS 感染了 5 个红辣椒种质（L085、L099、L228、L252 和 L265），适合在生长室中长期维持。受TRV感染的植物定期浇水施肥，直至果实成熟，渗透后需要14-16周。结果表明，97% 和 100% 的 pTRV2-C2b-CaCCS 感染的 L085 和 L228 植物产生黄色果实，而感染 pTRV2-C2b 的植物没有产生黄色果实，表明CaCCS在果实中的高效沉默（图 1g）。敲除 CaCCS通过 RT-PCR 证实了黄色果实中 TRV 感染的转录水平和持续性（图 1d，h）。这些结果表明，优化的 TRV 载体和选择的辣椒种质可以构成研究辣椒果实基因功能的有效 VIGS 系统。

总之，我们的研究优化了辣椒中基于 TRV 的 VIGS 方法，并提供了高效的载体和辣椒种质，可以促进整个辣椒生命周期的基因功能研究。该策略也可以应用于其他工厂的VIGS系统。