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Author: search.filters.author.Choudhary, NandlalSubject: search.filters.subject.Indian citrus ringspot virus (ICRSV)

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    Identification of an RNA silencing suppressor encoded by an Indian citrus ringspot virus
    (Springer Nature Publishing AG, 2025) Angira, Aniket; Baranwal, V. K.; Ranjan, Aashish; Choudhary, Nandlal
    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 signifcant 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 Agrobacterium tumefaciens and agro-infltration into leaves of Nicotiana benthamiana 16c line. Only the leaves infltrated with 35s-GFP/ORF5 showed a GFP fuorescence 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 fuorescence signal. This fnding was further supported by beta-glucuronidase (GUS) histochemical assays by infltrating the construct expressing ORF5-GUS under 35s promoter in the leaves of N. benthamiana. Leaves infltrated with 35s-GUS/ORF5 formed diX-indigo precipitate similar to leaves infltrated 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-infltrated leaves. Together, these results suggest that ORF5 encoded protein has the potential RSS function of ICRSV which successfully suppresses host RNAi silencing mechanism.
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    Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India
    (Elsevier B.V., 2024) Angira, Aniket; Baranwal, V K; Ranjan, Aashish; Choudhary, Nandlal
    Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507bp partial coat protein gene (pCPG) segment was amplified from infected kinnow tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL-1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.