In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Abstract

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plantvector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

“…However, in this case, they were ground in 0.5 M tris buffer, pH 6.8 at 1:7 ratio (w/v). The procedure was carried out as described by Calegario et al (2012) using the CiLV-C p29 antibody diluted at 1:1000 (v/v).…”

“…Because CiLV-C virions have been elusive to purify probably due to their labile nature, a strategy would be to express viral proteins in vitro and produce antibodies, Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C... either mono-or polyclonal, after their purification. An attempt to express the MP (ORF 3, RNA 2) of CiLV-C and produce antibody was successful, but possibly due to the distinct folding pattern of the expressed protein, this antibody reacted poorly with native MP and was not suitable for immunoassays (Calegario et al, 2012). Another approach was then attempted, expressing the p29 protein, a structural putative coat protein of the virion (ORF 2, RNA 1).…”

Development of polyclonal antiserum against movement protein from Potato leafroll virus and its application for the virus detection

“…Most plant viruses contain MP genes in their genome that are associated with cell-to-cell movement of the virus through the plasmodesmata (Wolf et al 1989;Haupt et al 2005;Akamatsu et al 2007). Anti-MP antisera used to detect these MPs in different plant viruses have been reported (Xie et al 2007;Calegario et al 2012;Li et al 2015;Koolivand et al 2016). As far as we know, there is no report on the preparation of PLRV-MP antiserum used for the detection of PLRV and its MP.…”

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

“…Most of the studies on virus MPs are focused on their function or loss of function through mutations, production of transgenic MP-expressing resistant plants, cellular localization, increase in the size exclusion limit of plasmodesmata, and interactions between MP and other cell proteins ( Akamatsu et al, 2007 ; Deom et al, 1990 ; Haupt et al, 2005 ; Wolf et al, 1989 ). Also, MPs as non-structural proteins have contributed to antibody production and in vivo virus detection ( Calegario et al, 2012 ; Cerovska et al, 2012 ; Salimi et al, 2010 ).…”