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Virology 263, 155–165 (1999) Article ID viro.1999.9901, available online at http://www.idealibrary.com on Nematode Transmission of Tobacco Rattle Virus Serves as a Bottleneck to Clear the Virus Population from Defective Interfering RNAs Peter B. Visser,* Derek J. F. Brown,† Frans Th. Brederode,* and John F. Bol*,1 *Institute of Molecular Plant Sciences, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands; and †Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland Received April 26, 1999; returned to author for revision June 1, 1999; accepted July 12, 1999 DI7 is a defective interfering RNA derived from RNA 2 of tobacco rattle tobravirus (TRV) isolate PpK20. Tobacco was transformed with DI7 cDNA fused to the CaMV 35S promoter. Upon infection of the transgenic plants with TRV isolate PpK20 or the serologically unrelated isolate PaY4, the transgenic DI7 RNA started to accumulate at high levels and strongly interfered with accumulation of wild-type (wt) RNA 2. When DI7 transgenic plants infected with isolate PpK20 were used as source plants in nematode-transmission experiments, the vector Paratrichodorus pachydermus efficiently transmitted virus to healthy bait plants. However, the nematodes transmitted only the wt virus present in the transgenic source plants, whereas virus particles containing the abundant, accumulated DI7 RNA were excluded from transmission. Evidence is presented that wt RNA 2 and DI7 RNA are encapsidated in cis by their encoded CPs, which are known to be functional and nonfunctional in transmission, respectively. This mechanism would result in defective interfering RNAs, which rapidly arise after mechanical transmission of the virus in the laboratory, being eliminated from tobraviruses under natural field conditions. Also this mechanism which acts with nematode transmitted virus isolates contrasts with that of vector-transmission of defective potyviruses and luteoviruses by wt helper viruses. © 1999 Academic Press INTRODUCTION At present, seven nematode species within the genus Paratrichodorus and four within Trichodorus are known Tobacco rattle virus (TRV) is the type member of the to be vectors of tobraviruses (Brown et al., 1995). A highly genus Tobravirus, a group of plant viruses that are trans- specific relationship has been found between tobravirus mitted by root-feeding nematodes. The tobravirus ge- strains and nematode species involved in their transmis- nome consists of two positive-sense RNAs that are sep- arately encapsidated into rod-shaped particles. The sion. Serologically distinct isolates have been shown to longer genome segment, RNA 1, encodes the genes for be transmitted by different nematode species (Brown et replication and movement of the virus and a small pro- al., 1989; Ploeg et al., 1992). Recently cDNA copies of tein of unknown function and is highly conserved among RNA 2 from nematode transmissible isolates of TRV- TRV isolates (Harrison and Robinson, 1986). In contrast, PpK20 (Herna´ndez et al., 1995), TRV-TpO (MacFarlane et the smaller RNA 2 is highly variable among different al., 1999), and pea early browning virus (PEBV) isolate isolates in both nucleotide sequence and genome TPA56 were characterized (MacFarlane and Brown, length. In addition to the viral coat protein (CP), RNA 2 1995). Transmission studies carried out with various mu- may encode one or more nonstructural proteins depend- tants of these isolates provided genetic evidence that a ing on the isolate. It has been shown that some of these 29.4-kDa protein from TRV-PpK20 (Hernandez et al., proteins are involved in nematode transmission (Mac- 1997) and both the 29- and 23-kDa proteins, and possibly Farlane et al., 1996, 1999; Herna´ndez et al., 1997). The a 9-kDa protein from PEBV TPA56 (MacFarlane et al., unique distribution of genes over two genome segments 1996), are required for transmission of these viruses by permits the RNA 1 of tobraviruses to be infectious alone. their vector nematodes. Sequence similarity was found In this so-called nonmultiplying (NM) type of infection, between the 9- and 29-kDa proteins of TRV isolate TpO nonencapsidated RNA 1 is able to replicate and spread and PEBV isolate TPA56, which share the same vector throughout the plant, but because of the lack of genetic nematode. However, no significant sequence homology information encoded by RNA 2, the virus can not be was found with the 29.4-kDa protein of TRV isolate transmitted by vector nematodes (Harrison and Robin- PpK20, which is transmitted by another nematode spe- son, 1986). cies, suggesting that the nonstructural proteins are rel- evant to the specificity of nematode transmission (Mac- Farlane et al., 1999). Although the molecular mechanism 1 To whom reprint requests should be addressed. Fax: 131 71 underlying the transmission process is still unknown, it is 5274340. E-mail: [email protected]. suggested that the nonstructural proteins might specifi- 0042-6822/99 $30.00 155 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 156 VISSER ET AL. cally link the virus particles to receptor sites in the nematode oesophagous (Herna´ndez et al., 1997). The proteins then could be considered as helper compo- nents, functionally similar to the helper component of potyviruses or the aphid transmission factor of caulimo- viruses (Pirone and Blanc, 1996). Tobacco rattle virus is an economically important virus causing disease in potato crops and bulbous ornamen- tals. Currently disease control is achieved by applying FIG. 1. Schematic representation of RNA 2 of TRV isolate PpK20 and highly toxic soil sterilant chemicals, which reduce or the defective RNA DI7. Regions homologous between RNA 1 and RNA eliminate nematode populations, and oximacarbamates, 2 are indicated by a solid bar; RNA-2-specific sequences are indicated which act as nematastats that temporarily immobilise the by an open bar. The relative positions of the ORFs are given (numbers indicate the molecular masses in kilodaltons of the potential protein nematodes, stopping them from moving or feeding, thus products). The mutated coat protein encoded by DI7 RNA has been preventing them from transmitting virus. As an alternative designated mCP. The dotted line in DI7 RNA represents the deleted to the use of these toxic agro-chemicals, attempts have region with respect to RNA 2. been made to obtain genetically engineered resistance of plants to tobravirus infection. Tobacco plants trans- formed with the CP gene of TRV isolates TCM or PLB nematode Paratrichodorus pachydermus exclusively were highly resistant to mechanical infection (Van Dun transmitted virus particles containing wt RNA 2 but not and Bol, 1988; Angenent et al., 1990). However, it was particles containing the DI7 RNA to healthy bait plants. shown that this type of CP-mediated resistance was not Evidence is presented that encapsidation in cis of RNA 2 effective against virus transmitted by vector nematodes and DI7 RNA by their encoded CPs provides the mech- (Ploeg et al., 1993). Moreover, CP-mediated resistance to anism used by TRV to eliminate DI RNAs from the virus TRV is limited to strains carrying a CP gene that is population during transmission by the natural vector. homologous to the transgene and will therefore not be effective against the wide variety of TRV serotypes that RESULTS are found in the field. In another approach, plants were Generation of DI7 transgenic plants transformed with part of the 201-kDa replicase gene from PEBV RNA 1 of isolate SP5. Although a broad-range In our studies of TRV, we noticed an error in the resistance was expected because of substantial se- sequence of RNA 2 of TRV isolate PpK20 published by quence homology of replicase genes among different Herna´ndez et al., (1995). The presence of the U residue tobraviruses, the transformed plants were only resistant at position 2106 of the published sequence was not to inoculation with two PEBV isolates and not to TRV or confirmed in the present study, and the deletion of this pepper ringspot tobravirus (MacFarlane and Davies, residue results in an extension of the reading frame of 1992). the 29.4-kDa protein to that of a 97 amino acids (aa) In this study, we have explored the possibility of inter- longer protein of 40.0 kDa. The revised genome structure fering with vector transmission of TRV isolates by trans- of PpK20 RNA 2 is shown in Fig. 1 and the sequence genic expression of a defective interfering (DI) RNA. DI deposited in GenBank (Accession No. Z36974) has been RNAs arise rapidly when TRV is repeatedly transferred corrected. from plant to plant by mechanical inoculation. It was The structure of DI RNA DI7 (D7 described by Herna´n- previously shown that DI7 RNA, a DI RNA derived from dez et al., 1996) is shown in Fig. 1. This DI RNA contains RNA 2 of TRV isolate PpK20, completely outcompeted the 59-terminal 1126 nucleotides (nt) and the 39-terminal wild-type (wt) RNA 2 in a mixed infection. This DI RNA 895 nt of RNA 2 joined by a sequence of 18 nt. DI7 encodes a C-terminally truncated CP but no nonstruc- encodes a mutant CP (mCP) of which the C-terminal 15 tural proteins. DI7 RNA coreplicated with RNA 1, and both aa of wt CP are replaced by the sequence LRL. In RNAs were encapsidated by the DI-encoded CP, but the addition, the 40K and 32.8K genes have been completely progeny virus was no longer transmitted by the nema- and partially deleted, respectively, in DI7. mCP was func- tode vector (Herna´ndez et al., 1996). Here we report that tional in encapsidation of viral RNAs but defective in DI7 RNA expressed from a nuclear DNA copy in trans- nematode transmissibility (Herna´ndez et al., 1996; Mac- genic tobacco plants also coreplicated with RNA 1 and is Farlane et al., 1996). To generate transgenic tobacco able to compete with the replication of wild-type RNA 2 of expressing DI7, the sequence containing the CaMV 35S two serologically distinct tobraviruses.

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