In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

中文翻译：

---

RNA沉默相关基因有助于易感番茄植株对马铃薯病毒X和Y感染的耐受性

在植物中，RNA 沉默系统在其被病毒衍生的双链 RNA 诱导后起到抗病毒防御机制的作用。这是通过 RNA 沉默成分的作用发生的，包括 Dicer 样 (DCL) 核酸酶、Argonaute (AGO) 蛋白和 RNA 依赖性 RNA 聚合酶 (RDR)。植物编码多个 AGO、DCL 和 RDR。这些成分的功能主要在拟南芥和本氏烟草中进行了研究。在这项研究中，我们研究了 DCL2、DCL4、AGO2、AGO3 和 RDR6 在番茄对病毒感染的反应中的作用。为此，我们使用了转基因番茄植物（Solanum lycopersicum cv. Moneymaker），其中这些基因的表达被双链 RNA 介导的 RNA 沉默抑制。我们之前创建了多个 DCL（即 DCL2 和 DCL4）（hpDCL2. 4) 和 RDR6 (hpRDR6) 敲低转基因番茄植物，这里还做了多个 AGO（即 AGO2 和 AGO3）敲低植物（hpAGO2.3），其中表达与这些基因同源的双链 RNA 以诱导对它们的 RNA 沉默. 将马铃薯病毒 X (PVX) 和 Y (PVY) 接种到这些转基因番茄植株上，并研究了这些植株对病毒的反应。除了观察症状外，还通过蛋白质印迹和北印迹以及逆转录聚合酶链反应 (RT-PCR) 检测了病毒外壳蛋白和基因组 RNA。通过定量RT-PCR研究宿主mRNA水平。接种 PVX 后，与其他接种的植物相比，hpDCL2.4 植物出现了更严重的系统性花叶，并伴有卷叶。在hpAGO2.3植物中也观察到系统性坏死。尽管症状严重程度不同，但未接种的上部叶片中 PVX 外壳蛋白 (CP) 和基因组 RNA 的积累在 hpDCL2.4、hpRDR6 和 hpAGO2.3 植物与空载体转化植物之间没有明显差异。赚钱的番茄植株在感染 PVY 后无症状。然而，接种 PVY 的 hpDCL2.4 植物出现了症状，包括卷叶。一致地，通过蛋白质印迹在未接种的 hpDCL2.4 植物有症状的上部叶片中检测到 PVY CP。值得注意的是，在其他无症状的转基因或野生型植物中很少检测到 PVY CP。然而，使用逆转录聚合酶链反应在所有接种植物的未接种上部叶片中检测到 PVY。这些发现表明 PVY 以低水平系统感染无症状的 Moneymaker 番茄植株（即，通过蛋白质印迹未检测到 CP）。我们的结果表明，番茄品种 Moneymaker 对 PVX 敏感并显示出轻微的花叶症状，而它对系统性 PVY 感染具有低病毒滴度的耐受性和无症状。相比之下，在 hpDCL2.4 植物中，PVX 诱导的症状变得更加严重，PVY 感染引起了症状。这些结果表明 DCL2、DCL4 或两者都有助于对 PVX 和 PVY 感染的耐受性。PVY CP 和基因组 RNA 在 DCL2.4 击倒植物中积累的程度更大。因此，这些 DCL 对 PVY 感染耐受性的贡献至少部分归因于它们在抗病毒 RNA 沉默中的作用，其控制了番茄植物中 PVY 的繁殖。