Angira Aniket, Baranwal V K, Ranjan Aashish, Choudhary Nandlal
Amity Institute of Virology & Immunology, Amity University Uttar Pradesh, Noida, Uttar Pradesh 201313 India.
Division of Plant Pathology, Advanced Centre of Plant Virology, Indian Agricultural Research Institute, Pusa, New Delhi, 110012 India.
Physiol Mol Biol Plants. 2025 Jan;31(1):93-104. doi: 10.1007/s12298-024-01524-8. Epub 2024 Nov 9.
Plant viruses encode RNA silencing suppressor (RSS) proteins to counter the induced antiviral defense, an RNAi silencing mechanism of the host. Indian citrus ringspot virus (ICRSV) causes the ringspot disease, which leads to significant yield loss of kinnow orange. The ICRSV genome contains six open reading frames (ORFs), however, the ORF encoding the potential RSS is not yet known. In this study, we have attempted to identify the RSS protein of ICRSV. To this end, ORF 2,3,4,5 and 6 were cloned into pCAMBIA1302 (35s-GFP) vector, followed by transformation of and agro-infiltration into leaves of 16c line. Only the leaves infiltrated with 35s-GFP/ORF5 showed a GFP fluorescence signal similar to 35s-GFP/P19, a well-studied positive RSS. Usually, the induced host RNAi silencing is supposed to cleave the expressed GFP-RNA. However, it is suspected that ORF5-encoded protein was able to suppress the host silencing mechanism, leading to the retention of the GFP fluorescence signal. This finding was further supported by beta-glucuronidase (GUS) histochemical assays by infiltrating the construct expressing ORF5-GUS under 35s promoter in the leaves of . Leaves infiltrated with 35s-GUS/ORF5 formed diX-indigo precipitate similar to leaves infiltrated with, indicating the RSS activity of ICRSV. Later, semi-quantitative PCR and quantitative reverse transcription PCR qRT-PCR) assays showed a higher expression of GFP and GUS in ORF5 agro-infiltrated leaves. Together, these results suggest that ORF5 encoded protein has the potential RSS function of ICRSV which successfully suppresses host RNAi silencing mechanism.
植物病毒编码RNA沉默抑制子（RSS）蛋白以对抗宿主诱导的抗病毒防御，这是一种RNA干扰沉默机制。印度柑橘环斑病毒（ICRSV）引发环斑病，导致金诺橘产量大幅损失。ICRSV基因组包含六个开放阅读框（ORF），然而，编码潜在RSS的ORF尚不清楚。在本研究中，我们试图鉴定ICRSV的RSS蛋白。为此，将ORF 2、3、4、5和6克隆到pCAMBIA1302（35s-GFP）载体中，随后转化并农杆菌渗入16c系叶片。只有用35s-GFP/ORF5渗入的叶片显示出与35s-GFP/P19相似的绿色荧光蛋白（GFP）荧光信号，35s-GFP/P19是一种经过充分研究的阳性RSS。通常，诱导的宿主RNA干扰沉默应该切割表达的GFP-RNA。然而，怀疑ORF5编码的蛋白能够抑制宿主沉默机制，导致GFP荧光信号保留。通过在叶片中渗入在35s启动子下表达ORF5-GUS的构建体进行的β-葡萄糖醛酸酶（GUS）组织化学分析进一步支持了这一发现。用35s-GUS/ORF5渗入的叶片形成了与用……渗入的叶片相似的二氯靛蓝沉淀，表明ICRSV的RSS活性。后来，半定量PCR和定量逆转录PCR（qRT-PCR）分析显示，在ORF5农杆菌渗入的叶片中GFP和GUS表达较高。总之，这些结果表明，ORF5编码的蛋白具有ICRSV的潜在RSS功能，成功抑制了宿主RNA干扰沉默机制。
