In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus

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Highlights

  • CTV p23 gene was cloned and polyclonal antibodies raised against bacterially expressed recombinant CTV p23 protein.

  • Produced polyclonal antibodies were used to develop a sensitive and economically affordable CTV detection tool.

  • RT-PCR assay using CTV p23 gene specific primers was 1000 times more sensitive compared to coat protein gene based primers.

  • In silico characterizations of CTV p23 protein has been accomplished.

Abstract

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Introduction

Citrus is the third most important fruit crop in India after banana and mango. It contributes significantly to the agricultural economy of the country. It is also the most important fruit crop worldwide, presently grown in more than 150 countries. It is originated in the tropical and sub-tropical regions of south-east Asia [1]. Recent data suggests an estimated citrus production of 11.7 million metric ton (MT) from an area of 0.97 million hectares (ha) in India [2]. Although India ranks fourth in the production of citrus fruit worldwide, the average citrus productivity is 12.3 MT/ha which is far below than the average productivity of 28–35 MT/ha obtained in the frontline citrus growing countries. The drastic difference in the yield is potentially attributed to the biotic stresses caused mainly by viruses and virus-like diseases and lack of their management practices [1,3]. Citrus tristeza disease, caused by citrus tristeza virus (CTV) is regarded as the most damaging and destructive for the citrus that causes huge economic loss with total dieback or decline of citrus trees [4,5]. CTV belongs to the genus Closterovirus, family Closteroviridae and occurs in all regions of the world where citrus is grown [[6], [7], [8]]. The virions of CTV are filamentous particle of 2000 × 11 nm. They contain an approximately 20 kb positive sense ssRNA genome with 12 open reading frames (ORFs) encoding at least 19 different proteins [[9], [10], [11]]. CTV infects all the species, cultivars, and hybrids of citrus and to date, there are no reports of resistant cultivars or any therapeutic compounds against the virus [[10], [11], [12]].

The virus is transmitted by budding and grafting in the nursery and by number of aphid vectors e.g., Toxoptera citricida Kirkaldy, Aphis gossypii Glover and Aphis spiraecola Patch in the groves [13]. CTV infection induces different symptoms based on citrus species, rootstock-scion combination, virus strain, and environmental conditions that includes stem pitting, vein clearing, vein flecking, stunting, slow decline, and quick decline in the field trees [7]. The biological indexing is one of the best methods for CTV detection [14,15]. The virus can also be detected by ELISA, electron microscopy, dot immunobinding assay and immuno electron microscopy [1,16]. Currently, different advanced techniques have been reported for early detection of CTV viz., RT-LAMP, IC-RT-LAMP, coat protein based immunoassay and electronic nose system [[17], [18], [19], [20], [21]]. However, the reverse transcription-polymerase chain reaction (RT-PCR) based method using coat protein gene specific primer set is the mostly used CTV detection technique [[22], [23], [24]]. The recent efforts are focusing on the development of a simple, rapid and improved CTV diagnostic technology.

RNA binding protein gene (CTV-p23) is located towards 3′-terminal and 5′ to the untranslated region (UTR) of the RNA genome of CTV. It encodes an ~23 kDa protein of 209 amino acids residues which plays an important role in the asymmetrical accumulation of CTV RNA [7,[25], [26], [27]]. It is known that subgenomic RNA (sgRNA) of CTV-p23 gene accumulates to a much higher levels compared to other sgRNAs and is considered a potential target for developing diagnostics [16]. It is also reported that CTV-p23 protein has the highest expression and strongest suppression activity among the all VSRs (viral suppressor of RNA silencing) [28]. The key amino acid residues that involved in the regulation of expression and silencing suppressor activity of CTV-p23 protein has been identified from a mild strain of CTV [28]. This is an important step towards understanding the viral pathogenecity and protein (antigen) level accumulation for early diagnosis of virus. In the present study, the CTV-p23 gene was amplified cloned and a polyclonal antibody was raised against bacterial expressed and purified recombinant protein with the objective of developing a suitable immunoassay, Western blot and ELISA for early virus detection. Further, in silico characterization of CTV-p23 protein was carried out to know the key amino acid residues involved in protein stability, expression level and silencing suppressor activity.

Section snippets

Sample collection and establishment of CTV isolates

Leaf and young twigs from suspected CTV infected citrus trees were collected from six different regions in India i.e., Vidharbha (Maharashtra), Marathwada (Maharashtra), Tripura, Assam, Prayagraj (Allahabad, Uttar Pradesh) and Meghalaya, for RT-PCR detection, biological indexing and establishment of CTV cultures/isolates. Representative CTV isolates of six geographic regions viz. AK1 (Khasi mandarin (Citrus reticulata), Assam), M5 (Mosambi (Citrus sinensis), Vidarbha), NRCC10 (Acid lime (Citrus

Cloning, expression and purification of CTV-p23

The RT-PCR assays showed high CTV incidence (25–50%) in the samples collected from different geographical regions of India (Table 2). It was also observed that indexing based on CTV-p23 primers (p23C2-RBP-F/R) recorded greater numbers of samples positive for CTV as compared to coat protein gene based primers (CN150/151) (Table 2). The six representative CTV isolates showing an expected ~672 bp amplicons using CN150/151 primers were separated on agarose gel (Fig. 1A). Further, in all the six

Discussion

During the last few decades, the tristeza disease has received international importance owing to its devastating impact on citrus crop yield because there is no known commercial source of resistance available in any cultivar of citrus so far [12]. It is believed that the destructive impact of CTV can be reduced by accurate detection and subsequent eradication of infected plants. There are reports for production of recombinant polyclonal antibody against the CTV-p25 protein and its utilization

CRediT author contribution statement

Sunil B. Kokane: Conceptualization, Investigation, Writing - original draft, preparation, Methodology, Visualization. Amol D. Kokane: Methodology, Visualization. Pragati Misra: Supervision, Formal analysis, Data curation. Ashish J. Warghane: Conceptualization, Investigation, Methodology. Pranav Kumar: Methodology, Formal analysis. Mrugendra G. Gubyad: Methodology, Visualization. Ashwani Kumar Sharma: Formal analysis, Resources. Kajal Kumar Biswas: Formal analysis. Dilip Kumar Ghosh:

Acknowledgements

Authors thank Dr. Siddarame Gowda, CREC, University of Florida, USA for reviewing this manuscript.

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