Production of Polyclonal Antibodies to a Recombinant Potato Mop-top Virus Non-structural Triple Gene Block Protein 1

The gene encoding the triple gene block protein 1 (TGBp1) of Potato mop-top virus (PMTV) was cloned into expression vector pQE32 tagging the protein with 6xHis on the N-terminus. When the gene was enshortened on its 3¢-end by two different restriction digestions, efficient and high yield bacterial expression was achieved in both cases, as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. One of these two constructs was used for raising rabbit polyclonal antibodies. The obtained sera and antibodies were tested for the detection of PMTV in laboratory host Nicotiana benthamiana and natural host Solanum tuberosum by enzyme-linked immunosorbent assay (ELISA) as well as by Western blots. The obtained antisera are more suitable for Western blot analysis of infected plants than for ELISA. Introduction The Potato mop-top virus (PMTV) is a fungus-transmitted soil-borne virus and the type member of the genus Pomovirus (Torrance and Mayo, 1997). PMTV occurs in potato-growing regions in Europe, North and South America and Asia in cool wet climate causing a wide range of symptoms in haulms and tubers, which vary depending on the potato cultivar and environmental conditions, thus complicating the identification of the virus disease (Jones, 1988). PMTV has tubular and rigid particles, measuring 18–22 · 100–150 or 250–300 nm (White et al., 1972) encapsidating three RNA components, namely RNA 1, RNA 2 and RNA 3 (Kallender et al., 1990; Kashiwazaki et al., 1995; Savenkov et al., 1999). The third RNA, 2.9 kb long, contains a triple gene block (TGB) encoding three proteins involved in cell-to-cell movement, and an additional open-reading frame (ORF) for a predicted cystein-rich protein (CRP) with unknown function (Mayo et al., 1996; Fig. 1). The analysis of nucleotide sequences showed that PMTV genomes from different parts of the world are highly conserved which is the fact that usually makes the diagnosing of viral disease more simple (Mayo et al., 1996; Sandgren et al., 2001; Nielsen and Nicolaisen, 2003; Čeřovská et al., 2003a; Pečenková et al., 2004). However, the detection based solely on the presence of PMTV coat protein (CP) seems to be insufficient as it was recently shown that the distribution of PMTV RNAs varies in different parts of infected plants and that the multipartite virus PMTV is capable of establishing infection without the CP-encoding RNA, and also without the putative CRP (Savenkov et al., 2003). Thus, detection based on some other non-structural protein could be advantageous when combined with CP detection methods and polymerase chain reaction (PCR) methods (Arif et al., 1994; Rantanen et al., 1999; Mumford et al., 2000). Antisera raised against triple gene block protein 1 (TGBp1), for example, could be used for such a purpose as well as for studying the fate of this protein in infected plant and its role in the viral movement through plant tissue. TGB proteins are found in viruses belonging to several different genera. The TGB1 proteins could be generally divided into two groups: Hordeivirus-like TGBp1 and Potexvirus-like TGBp1 (Morozov and Solovyev, 2003). In both cases TGBp1s contain a NTP/helicase sequence domain and belong to helicases of superfamily I (SF-I) with the typical presence of seven conserved motifs (Gorbalenya et al., 1989). Both hordei-like and potex-like TGBp1, like many known helicases, can form homodimers or oligomers and are capable of self-interaction (Gorbalenya and Koonin, 1993; Cowan et al., 2002). TGBp1 of rod-shaped Pomovirus and Hordeivirus are larger comparing with potex-like TGBp1s – 39–63 kDa and contain additional long N-terminal domains (Solovyev et al., 1999). The presence of arginine/lysine-rich clusters was found in these extensions as well as the region of sequence similarity upstream of the helicase domain (Solovyev et al., 1999). www.blackwell-synergy.com J. Phytopathology 154, 422–427 (2006) 2006 The Authors Journal compilation 2006 Blackwell Verlag, Berlin