Development of polyclonal antisera against movement proteins from three poleroviruses infecting cucurbits
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RESEARCH
Phytopathology Research
Open Access
Development of polyclonal antisera against movement proteins from three poleroviruses infecting cucurbits Shao-Kang Zhang1,2†, Tian-Yu Zhao1,2†, Xing Shi1,2†, Yu-Zi Liu1,2, Ying Wang1,2, Zong-Ying Zhang1,2, Da-Wei Li1,3, Jia-Lin Yu1,3, Qiao-Xia Shang4 and Cheng-Gui Han1,2*
Abstract Cucurbit aphid-borne yellows virus (CABYV), melon aphid-borne yellows virus (MABYV) and suakwa aphid-borne yellows virus (SABYV) are three poleroviruses that infect cucurbit crops. Developing specific antisera against such viruses is crucial for their detection and functional understanding of related genes. However, no studies have yet reported viral detection using antisera against movement proteins (MP) in these three viruses. In this study, we generated plasmids expressing three viral MP genes, and transformed them into the Escherichia coli strain, Rosetta, to recombinantly express and purify fusion proteins. Then, polyclonal antisera were derived by immunizing New Zealand white rabbits, after which western blotting was used to determine the titer, sensitivity and specificity of the antisera. The antisera titers against MPCABYV, MPMABYV and MPSABYV were 1:512000, 1:256000 and 1:256000, respectively. The optimized working concentrations for the three antisera ranged between 1:10000 and 1:64000. Additionally, antisera against MPCABYV and MPMABYV only reacted with their corresponding MP proteins. Antiserum against MPSABYV not only had the strongest reaction with its MP, but also reacted weakly with MPCABYV and MPMABYV. All three antisera exerted no serological reactions with other poleroviruses. Furthermore, our data showed that all antisera specifically detected MPs in both Nicotiana benthamiana and cucumber leaves. Thus, we have established a system that sensitively detects three poleroviruses infecting cucurbits, using antisera against MPs. We provide a foundation for future research on the serological detection of these viruses, and interaction mechanisms between viruses and host plants. Keywords: Cucurbit aphid-borne yellows virus, Melon aphid-borne yellows virus, Suakwa aphid-borne yellows virus, Movement protein, Prokaryotic expression, Antiserum preparation, Virus detection
Background Cucurbits are important fruit crops across the world. Plant viruses that infect cucurbits cause significant economic losses and limit cucurbits production (Gholamalizadeh et al. 2008; Knierim et al. 2014; Chikh-Ali et al. 2019). Cucurbit aphid-borne yellows virus (CABYV), * Correspondence: [email protected] † Shao-Kang Zhang, Tian-Yu Zhao and Xing Shi contributed equally to this work. 1 Ministry of Agriculture Key Laboratory of Pest Monitoring and Green Management, and State Key Laboratory for Agro-biotechnology, China Agricultural University, Beijing 100193, People’s Republic of China 2 Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, People’s Republic of China Full list of author information is available at the end of the article
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