Virology 254, 105–114 (1999) Article ID viro.1998.9527, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Satellite RNA Is Essential for Encapsidation of Groundnut Rosette Umbravirus RNA by Groundnut Rosette Assistor Luteovirus Coat Protein D. J. Robinson, E. V. Ryabov, S. K. Raj,1 I. M. Roberts, and M. E. Taliansky2 Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom Received September 11, 1998; returned to author for revision September 28, 1998; accepted November 17, 1998 Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette umbravirus (GRV), GRV satellite RNA (sat-RNA) and groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAV and GRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomic and satellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomic and satellite RNAs efficiently, giving a high yield of transcap- sidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CP and its capacity to promote aphid transmission of GRV was observed. © 1999 Academic Press INTRODUCTION tor virus (GRAV) (Casper et al., 1983; Reddy et al., 1985; Rajeshwari et al., 1987), but neither GRAV nor GRV indi- Rosette disease of groundnut occurs in Africa, south of vidually induce the symptoms of groundnut rosette dis- the Sahara, and causes severe crop damage. The dis- ease (Hull and Adams, 1968; Reddy et al., 1985). The ease is caused by a complex of agents comprising two major cause of the symptoms of the disease is GRV viruses and a satellite RNA (sat-RNA). Groundnut rosette sat-RNA (Murant et al., 1988; Murant and Kumar, 1990). virus (GRV) is a member of the genus Umbravirus (Mu- This is a single-stranded RNA of 895–903 nucleotides rant et al., 1995). The plant viruses in this group have that relies on GRV for its replication and that, more single-stranded RNA genomes but do not produce con- unusually, is needed (together with GRAV) for GRV to be ventional virus particles. They are mechanically trans- aphid-transmissible (Murant, 1990). Thus this RNA is missible, but each depends on a helper virus from the essential for the survival of GRV in nature. However, for family Luteoviridae for transmission by aphids (Murant et the sake of simplicity, we refer to it in this paper as GRV al., 1995; Taliansky et al., 1996). The entire nucleotide sat-RNA. Although different GRV sat-RNA variants con- sequence of GRV comprises 4019 nucleotides and con- tain up to five potential ORFs in either positive or nega- tains four open reading frames (ORFs) (Taliansky et al., tive sense (Blok et al., 1994), none of the ORFs is essen- 1996). Two ORFs at the 59 end of the RNA are expressed tial for replication of the sat-RNA or its spread in infected by a 21 frameshift to give a single protein, which ap- plants (Taliansky and Robinson, 1997). Likewise, the pro- pears to be an RNA-dependent RNA polymerase. The duction of symptoms in infected Nicotiana benthamiana other two ORFs overlap each other in different reading plants does not require any of the potential translation frames. The 27-kDa ORF3 protein seems to have a role in products but instead involves two untranslated elements long-distance virus movement in the infected plant (E. V. in the sat-RNA, which can act in trans (Taliansky and Ryabov, D. J. Robinson and M. E. Taliansky, manuscript in Robinson, 1997). preparation), but it is apparently not a coat protein (CP) The mechanism of transmission of the entire virus because GRV does not form virus particles. The 28-kDa complex responsible for rosette disease by the aphid ORF4 protein is a cell-to-cell movement protein (Talian- Aphis craccivora as well as the role of GRAV and GRV sky et al., 1996; Ryabov et al., 1998). For aphid transmis- sat-RNA in this process remains unclear. However, it is sion of GRV, the helper virus is groundnut rosette assis- suggested that genomic and satellite RNA molecules of GRV may be encapsidated by GRAV CP in a manner analogous to the encapsidation of carrot mottle umbra- 1 Present address: Plant Virus Laboratory, National Botanical Re- search Institute, Rana Pratap Marg, P.B.No 436 Lucknow-226001, India. virus RNA by the CP of its helper, carrot red leaf virus 2 To whom reprint requests should be addressed. Fax: 44–1382- (Waterhouse and Murant, 1983). The process of encap- 562426. E-mail address: [email protected]. sidation is therefore of considerable interest. However, it 0042-6822/99 $30.00 105 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 106 ROBINSON ET AL. is very difficult to study this process in groundnut plants, toms at the same time as nontransformed plants. For line the natural host of the virus complex, because of the low GRAV5, most plants developed more severe symptoms: yield of nucleoprotein particles in preparations purified in addition to showing the yellow blotch mosaic, the from this host plant. On the other hand, N. benthamiana, systemically infected leaves also became rugose and an experimental host for GRV and GRV sat-RNA, cannot deformed (Fig. 1). Immunosorbent electron microscopy be infected with GRAV. Indeed, no convenient experi- (ISEM) of the extracts from systemically infected leaves mental host for the entire complex is known. As an on grids coated with polyclonal antiserum against GRAV alternative approach, we generated transgenic N. revealed the presence of spherical luteovirus-like parti- benthamiana plants expressing the CP gene of GRAV. cles in all transgenic lines (see below), although the Although in natural infections luteovirus particles contain number of particles varied significantly between lines small amounts of a larger protein produced by occa- and was highest for line GRAV5. Virus-like particles were sional read-through of the major CP termination codon never detected in noninoculated transgenic plants or and required for aphid transmission (Gray and Banerjee, nontransformed N. benthamiana plants infected with the 1998), our transgenic plants did not contain the se- YB isolate of GRV. More importantly, virus-like particles quences coding for the read-through domain. Inoculation were not detected in plants of any of the transgenic lines of these plants with GRV isolates containing or lacking when they were infected with MC1, a satellite RNA-free sat-RNA showed that the sat-RNA is absolutely essential isolate of GRV, although MC1 RNA was encapsidated in for encapsidation of GRV RNA by GRAV CP and the the presence of an appropriate sat-RNA (see below). read-through domain is not required for this process. These results suggest that the GRAV CP expressed in transgenic plants is able to transcapsidate heterologous RESULTS (GRV genomic and satellite) viral but not cellular RNAs to Formation of virus particles in GRAV-CP transgenic give virus particles (hereafter referred to as transcapsi- plants inoculated with the YB isolate of GRV dated virus particles, TVP) and that sat-RNA might be essential for this process. Moreover, the read-through The gene encoding GRAV CP was inserted into trans- domain is not essential for such encapsidation. formation vector pROK2, a pBIN19 derivative (Bevan, 1984) between the cauliflower mosaic virus (CaMV) 35S Isolation, composition and properties of virus-like promoter and the nopaline synthase terminator, to give particles accumulated in GRAV CP transgenic plants the plasmid pROK2-GRAV CP. The resulting vector was infected with GRV-YB used for transformation of N. benthamiana plants. Twelve individual transgenic plants (primary transformants; T0 For further analysis of TVP formed in GRAV CP trans- generation), regenerated in the presence of kanamycin, genic plants inoculated with GRV-YB, line GRAV5 was se- were obtained. All of them were normal in appearance lected. In contrast to the phloem-limited character of luteo- and grew and developed like nontransformed plants. viral infections, it was expected that transgenic expression Total leaf RNA extracted from these plants was assayed of the GRAV CP gene might take place in different plant for GRAV CP gene transcripts by reverse transcription– tissues including mesophyll cells. Therefore for purification polymerase chain reaction (RT–PCR) using primers spe- of TVP from infected GRAV5 transgenic plants, a standard cific to the termini of the inserted CP gene sequences. All method of purification of luteoviruses (Takanami and Kubo, 12 primary transformants gave a product of the expected 1979) was employed but without the enzyme treatment size (;600 bp) for the CP gene sequence (data not steps used for digestion of cell walls in vascular tissues. shown). However, neither ELISA nor Western blot (immu- Yields of TVP were unexpectedly high, 2–4 mg/100 g of noblot) analysis with polyclonal antiserum or different fresh leaf material. A time course showing the yield of TVP monoclonal antibodies (Scott et al., 1996) prepared to in a typical experiment is presented in Fig. 2A. Electron GRAV detected the protein product of the CP transgene microscopy showed that all the TVP were essentially sim- in plant extracts.