Virology 263, 155–165 (1999) Article ID viro.1999.9901, available online at http://www.idealibrary.com on

Nematode Transmission of Tobacco Rattle 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 (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 -transmission experiments, the vector Paratrichodorus pachydermus efficiently transmitted virus to healthy bait plants. However, the 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 . © 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: ϩ31 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 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 5Ј-terminal 1126 nucleotides (nt) and the 3Ј-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. Upon infection of promoter, the DI7 cDNA, and the nos terminator was transgenic DI7 plants with TRV isolate PpK20, accumu- excised from the infectious clone of DI7 (pCaTD7) and lation of high levels of DI7 RNA in the leaves and roots of was inserted into the Agrobacterium tumefaciens trans- the plants was observed, but when these plants were formation vector pMOG800. This construct was used to used as source plants to feed nematodes, the vector transform Nicotiana tabacum cv. Samsun NN, and 12 TRANSMISSION OF TRV BY NEMATODES 157

FIG. 2. Accumulation of DI7 RNA in nine independent lines transformed with DI7 cDNA after mock inoculation (A) or inoculation with RNA 1 of TRV isolate PpK20 (B). The names of the transgenic lines are indicated on top of each panel. WT is a control loaded with total RNA extracted from nontransgenic plants inoculated with 10 ␮g/ml of purified particles of TRV isolate PpK20. The relative positions of wild-type RNA 2 and DI7 RNA are indicated in the left margin. Northern blots were loaded with 2 ␮g of total RNA extracted from a pooled sample of four plants of each transgenic line. Samples were taken from inoculated leaves, 5 days after infection. The blots were hybridized with a 32P-labeled probe corresponding to nucleotides 1–334 of RNA 2 that detects genomic RNA 2 and DI7 RNA. primary transformants were obtained. Although all lines nontransgenic plants accumulated wt RNA 2 (Fig. 3, showed a normal phenotype at the vegetative stage, only lanes 1–6). Conversely, the D7-6 and D7-7 plants accu- nine plants were able to set viable seed after self-polli- mulated relatively high levels of DI7 RNA, but the accu- nation and were used in further experiments. Seedlings mulation of wt RNA 2 was reduced to low or undetect- of these nine lines were all resistant to kanamycin, indi- able levels in several D7-7 plants (Fig. 3, lanes 13–18) cating that multiple copies of the transgene were inte- and particularly in D7-6 plants (Fig. 3, lanes 7–12). Ap- grated in the plant genome. When 12 plants of each line parently, the interference with RNA 2 accumulation of the were infected with TRV-PpK20, normal symptom devel- transgenically expressed DI7 RNA resembles that of DI7 opment was observed, suggesting that none of the lines RNA expressed from the infectious clone pCaTD7 (Her- had acquired a protein- or RNA-mediated resistance to na´ndez et al., 1996). TRV. As the migration rate of wt CP and the DI7 encoded The ability of transgenic DI7 RNA to coreplicate with mCP differ in Western blot analysis, interference by DI7 TRV RNA 1 was investigated by comparing RNA levels in also could be analyzed at the protein level. Fig. 4A shows mock inoculated transgenic plants and plants inoculated the accumulation of mCP in six D7-6 plants (lanes 7–12) with TRV-PpK20 RNA 1. Northern blots were loaded with and six D7-7 plants (lanes 13–18) inoculated with TRV- equal amounts of RNA from mock-inoculated DI7 plants PpK20 RNA 1. Nontransgenic plants inoculated with RNA (Fig. 2A) or RNA 1-inoculated DI7 plants (Fig. 2B) and the 1, used as controls, did not accumulate CP (Fig. 4A, lanes autoradiograms were exposed for the same time. Trans- 1–6). Of the 12 transgenic plants tested, three plants did genic DI7 RNA in mock-inoculated plants is not visible in not accumulate mCP (Fig. 4A, lanes 8, 13, and 16), and it Fig. 2A but could be detected after a longer exposure of was checked that this lack of mCP accumulation was the blot (result not shown). Figure 2B shows that after due to unsuccessful infection of the plants with RNA 1 inoculation with RNA 1 the accumulation of DI7 RNA was rather than to a defect in the translation of DI7 RNA detectable in eight of the nine transgenic DI7 lines. In (result not shown). mCP migrated consistently as two several experiments, the level of DI7 accumulation var- bands in Western blot analysis. This phenomenon also is ied depending on the level of infection with RNA 1. Lines observed sometimes for wt CP (Angenent et al., 1989) D7-6 and D7-7 were selected for further experiments. and may reflect the existence of different conformations in the presence of SDS. Figure 4B shows the accumula- Interference of transgenic DI7 with replication of tion of CP in transgenic and nontransgenic plants inoc- mechanically inoculated virus ulated with virus particles containing TRV-PpK20 RNAs 1 To investigate whether the transgenic DI7 interfered and 2. The accumulation of wt CP was detectable in with replication of wt RNA 2, six plants each of lines D7-6 infected nontransgenic plants (Fig. 4B, lanes 1–6). The wt and D7-7 were inoculated with virus particles containing CP migrates more slowly than the minor band of mCP. RNAs 1 and 2 of TRV-PpK20. Six nontransgenic tobacco After infection of six D7-6 plants with the complete TRV- plants were similarly inoculated as a control. All of the PpK20 genome, the accumulation of CP resembles that 158 VISSER ET AL.

FIG. 3. Accumulation of wt RNA 2 and DI7 RNA in DI7 transgenic plants infected with TRV particles containing RNAs 1 and 2 of isolate PpK20. Six nontransgenic control plants (lanes 1–6) and six plants of the transgenic lines D7-6 (lanes 7–12) and D7-7 (lanes 13–18) were inoculated with TRV particles (10 ␮g/ml). Total RNA extracts (2 ␮g) of each plant were loaded on a Northern blot. Samples were taken from inoculated leaves, 5 days after infection. The blot was hybridized with a 32P-labeled probe corresponding to nucleotides 1–151 of RNA 2 that detects genomic RNA 2 and DI7 RNA. Lanes WT and DI7 contain RNA 2 and DI7 RNA as markers, respectively. The positions of RNA 2 and DI7 RNA are indicated in the left margin. in D7-6 plants inoculated with RNA 1 only: a major band mCP, which is defective in nematode-transmission, it of mCP can be seen without a clear detection of wt CP was possible that nematodes would be unable to ac- (Fig. 4B, lanes 7–12). mCP is also the major CP that quire transmissible virus from such source plants. More- accumulates in D7-7 plants inoculated with TRV-PpK20, over, when viruliferous nematodes transmit wt virus to but the increased intensity of the slower migrating band healthy DI7 bait plants, the transgenic DI7 RNA could indicates that in some of the plants minor amounts of wt render the infection nontransmissible by out competing CP are produced. Again, this indicates that interference the wt RNA 2. To investigate these possibilities, D7-6 and with RNA 2 replication in D7-6 plants is stronger than in D7-7 plants were used as source plants or bait plants in D7-7 plants. transmission experiments with P. pachydermus, the nat- To determine whether replication of transgenic DI7 is ural vector of TRV isolate PpK20. supported by heterologous TRV isolates, transgenic and Table 1 summarizes the data of two series of trans- nontransgenic plants were inoculated with virus parti- mission experiments. In Series 1, nematodes were given cles containing RNAs 1 and 2 of TRV isolate PaY4, which access to the root system of nontransgenic Nicotiana is serologically unrelated to isolate PpK20. Figure 5 clevelandii source plants, the leaves of which had been shows that six inoculated D7-6 plants each accumulated mechanically inoculated with TRV-PpK20. After a high levels of DI7 RNA (lanes 7–12), whereas five of six 4-weeks period, the nematodes were transferred to the inoculated D7-7 plants accumulated DI7 RNA (lanes 13– roots of transgenic D7-6 or D7-7 bait plants or nontrans- 18; infection of the plant analyzed in lane 17 was unsuc- genic tobacco bait plants. The roots of the source plants cessful). After inoculation of nontransgenic plants, no were tested to verify the presence of infectious virus by accumulation was observed of DI RNAs that may have inoculating root homogenates to indicator plants. The been present in the PaY4 preparation (Fig. 5, lanes 1–6). data in Table 1 (number of plants with virus in root Although the PaY4-encoded replicase efficiently repli- system/number of plants tested) confirm that all source cated the transgenic DI7 RNA, the results indicated that plants became infected. After the nematodes were given DI7 interfered less efficiently with the accumulation of a further period of 4 weeks to transmit virus to the bait PaY4 RNA 2 than with accumulation of the homologous plants, the roots of the bait plants were analyzed for the PpK20 RNA 2. presence of infectious virus. Transmission to transgenic or nontransgenic plants was successful in 90–100% of Effect of DI7 RNA on transmission of TRV by the assays (Table 1). This was expected because corep- nematodes lication of DI7 with wt virus in the transgenic bait plants The primary objective of our study was to determine would not affect the production of infectious units. To whether the transgenic DI7 RNA could render a TRV investigate this possible coreplication of DI7, RNA ex- infection nontransmissible by nematodes. As DI7 plants tracted from the roots of six plants from each of the three infected with TRV-PpK20 accumulate predominantly types of bait plants used in the experiments of Series 1 TRANSMISSION OF TRV BY NEMATODES 159

FIG. 4. Accumulation of wt coat protein (CP) and mutant coat protein (mCP encoded by DI7 RNA) in DI7 transgenic plants infected with RNA 1 or RNAs 1 and 2 of TRV isolate PpK20. Six nontransgenic control plants (lanes 1–6) and six plants of the transgenic lines D7-6 (lanes 7–12) and D7-7 (lanes 13–18) were inoculated with TRV RNA 1 (A) or particles containing TRV RNAs 1 and 2 (B). Proteins extracts from inoculated leaves were subjected to Western blot analysis, 5 days after infection. Lanes M were loaded with protein from mock-inoculated plants. The positions of wt CP and mCP are indicated in the left margin. was analyzed by Northern blot hybridization. Figure 6 prevent the nematodes from acquiring transmissible vi- shows that in the roots of nontransgenic bait plants only rus from the roots of these plants. wt RNA 2 is detectable (lanes 1–6). wt RNA 2 was An unexpected result was obtained when RNA ex- detectable in the roots of four of the six D7-6 bait plants tracted from the roots of source and bait plants from the (Fig. 6, lanes 7–12) and in all D7-7 bait plants (Fig. 6, experiments of Series 2 were analyzed by Northern blot lanes 13–18). In all infected bait plants, the transgenic hybridization. Figure 7A shows the RNAs in the roots of DI7 RNA had started to coreplicate with the transmitted the three types of source plants. As expected, the roots virus, but the interference with accumulation of wt RNA 2 of the nontransgenic source plants contained wt RNA 2 was less efficient than observed in leaves of DI7 plants and no DI RNA (Fig. 7A, lanes 1–6). In the roots of most that were mechanically inoculated with TRV-PpK20. of the D7-6 plants (Fig. 7A, lanes 7–12) and D7-7 plants In Series 2, transgenic D7-6 or D7-7, or nontrangenic (Fig. 7A, lanes 13–18), accumulation of wt RNA 2 was tobacco plants were used as source plants. Again, all reduced and DI7 RNA was the most abundant RNA-2- source plants inoculated with TRV-PpK20 were found to specific molecule. In the D7-7 plants analyzed in lanes 14 contain infectious virus in their root system (Table 1). and 18 of Fig. 7A, accumulation of wt RNA 2 was elimi- Nematodes recovered from these source plants were nated or reduced to below the sensitivity of the detection given access to the root system of nontransgenic N. method used. Figure 7B shows the accumulation of RNA clevelandii bait plants. As shown in Table 1, infectious in the roots of nontransgenic bait plants that were in- virus could be detected in the roots of all bait plants used fected by nematode-transmitted virus acquired from non- in the experiments of Series 2. Apparently the presence transgenic source plants (lanes 1–6), D7-6 source plants of DI7 RNA in the transgenic source plants did not (lanes 7–12) or D7-7 source plants (lanes 13–18). Due to 160 VISSER ET AL.

FIG. 5. Accumulation of wt RNA 2 and DI7 RNA in DI7 transgenic plants infected with particles containing RNAs 1 and 2 of TRV isolate PaY4. Six nontransgenic control plants (lanes 1–6) and six plants of transgenic lines D7-6 (lanes 7–12) and D7-7 (lanes 13–18) were inoculated with a leaf homogenate of plants infected with TRV isolate PaY4. Total RNA extracts (2 ␮g) from each plant were analyzed by Northern blot hybridization with a mixture of 32P-labeled probes corresponding to nucleotides 1–334 of PpK20 RNA 2 (for detection of DI7) and nucleotides 84–540 of PaY4 RNA 2 (for detection of PaY4 RNA 2). RNAs were extracted from inoculated leaves, five days after inoculation. Lanes WT and DI7 contain TRV-PpK20 RNA 2 and DI7 RNA as markers, respectively. The positions of RNA 2 and DI7 RNA are indicated in the left margin. the variable amounts of root tissue available, relatively was reduced below the detection level, such as in the weak signals were obtained in lanes 13 and 17 of Fig. 7B. plants analyzed in Fig. 7A, lanes 14 and 18. However, the results of Fig. 7B clearly demonstrate that irrespective whether the virus was acquired from trans- Encapsidation of RNA 2 and DI7 RNA in a mixed genic or nontransgenic source plants, the nematodes infection transmitted exclusively virus particles containing wt RNA 2 and not particles containing DI7 RNA although these Encapsidation in cis of RNA 2 and DI7 RNA by their were abundantly present in the roots of the transgenic encoded CPs could explain the exclusion of DI7 RNA source plants. It is concluded from the data in Table 1 from transmission by nematodes. To test this possibility, that in the experiments of Series 2 all bait plants were Nicotiana benthamiana plants were inoculated with a infected. This demonstrates that the vector nematodes mixture of TRV-PpK20 RNA 1 and cDNA clones of RNA 2 were able to selectively acquire wt virus even from trans- and DI7 RNA that were fused to the CaMV 35S promoter. genic source plants in which accumulation of wt RNA 2 The cDNA clones of RNA 2 and DI7 RNA were added to the inoculum in a 10:1 ratio, which results in the accu- mulation of similar amounts of RNA 2 and DI7 RNA in the TABLE 1 progeny virus (Herna´ndez et al., 1996). Virus isolated Transmission of TRV Isolate PpK20 by Paratrichodorus pachyder- from the plants was centrifuged in a sucrose gradient to mus from Nontransgenic Source Plants to Transgenic Bait Plants obtain partial separation of particles containing RNA 1, (Series 1) and from Transgenic Source Plants in Nontransgenic Bait RNA 2 and DI7 RNA. Figure 8 shows an analysis of the Plants (Series 2) RNAs (A, Northern blot) and proteins (B, Western blot) in Source plants Bait plants the fractions of the sucrose gradient. If DI7 RNA and RNAs 1 and 2 would be randomly encapsidated in trans Series 1 by wt CP and mCP expressed in the infected cells, all N. clevelandii 20/20a N. tabacum D7-6b 18/20 fractions of the gradient should contain similar amounts N. clevelandii 10/10 N. tabacum D7-7 10/10 of wt CP and mCP. However, Fig. 8 shows that mCP N. clevelandii 20 /20 N. tabacum control 19/20 largely cosediments with DI7 RNA, whereas wt CP co- Series 2 N. tabacum D7-6 20 /20 N. clevelandii 20/20 sediments with RNA 2, consistent with the hypothesis N. tabacum D7-7 20 /20 N. clevelandii 20/20 that the two RNAs are encapsidated in cis by their N. tabacum control 10 /10 N. clevelandii 10/10 encoded CPs. Compared with Fractions 3–6 of the gra- dient, the amount of mCP is reduced in Fractions 7–9 but a Values represent the number of plants with virus in the root system/ number of plants tested. increases again in Fractions 10–12, which contain major b Transgenic N. tabacum lines D7-6 and D7-7 express DI7 RNAs; amounts of RNA 1. Possibly, RNA 1 is encapsidated in untransformed N. tabacum plants were used as controls. trans by both type of CPs present in the mixed infection. TRANSMISSION OF TRV BY NEMATODES 161

FIG. 6. Accumulation of wt RNA 2 and DI7 RNA in roots of the bait plants used in the experiments of Series 1 in Table 1. Nematodes that had been given access to the roots of nontransgenic N. clevelandii source plants infected with TRV-PpK20, were transferred to nontransgenic tobacco bait plants (lanes 1–6) and DI7 transgenic bait plants of lines D7-6 (lanes 7–12) or D7-7 (lanes 13–18). After the nematodes were given a period of 4 weeks to transmit virus to the bait plants, RNA was extracted from the roots of the bait plants and analyzed by Northern blot hybridization. The blot was hybridized with a 32P-labeled probe corresponding to nucleotides 1–334 of TRV-PpK20 RNA 2. Lanes WT and DI7 contain RNA 2 and DI7 RNA as markers, respectively. The positions of RNA 2 and DI7 RNA are indicated in the left margin.

DISCUSSION mato bushy stunt virus was recently reported to confer broad-spectrum protection of the plants against tombus- Available evidence indicates that tobraviruses are viruses (Rubio et al., 1999). Our observation that trans- able to rapidly adapt the structure of their genome to a genic DI7 RNA did not interfere with transmission of change in selection pressure. Previously we have shown TRV-PpK20 by its vector nematode P. pachydermus dem- that after serial passage of the nematode-transmissible onstrated that the DI-based strategy will not control the TRV isolate PpK20 by mechanical inoculation, DI RNAs appear that have lost nonstructural genes from RNA 2 spread of tobraviruses under field conditions. However, that are required for nematode transmission when virus the reason for the failure of this strategy provided inter- particles are used as inoculum and also have shown that esting new information on the molecular basis of the the CP gene is lost from RNA 2 when phenol-extracted transmission process. It is well documented that tobra- RNA is used as inoculum in each passage (Herna´ndez et viruses are transmitted by their vector nematodes as al., 1996). This phenomenon may explain why many lab- virions: NM infections by RNA 1 are nontransmissible, oratory isolates of tobraviruses are no longer transmis- mutations in the CP gene can interfere with transmis- sible by vector nematodes. In the present study, we sion, and EM studies revealed the presence of TRV investigated the possibility of preventing transmission of particles at receptor sites in P. pachydermus (Brown et TRV by its vector nematodes by transgenic expression in al., 1995; MacFarlane et al., 1996). Moreover, EM studies plants of a DI RNA (DI7). The DI RNA (DI7) was derived revealed that there is sufficient space between the sur- from RNA 2 of TRV-PpK20 that had lost the nonstructural face of the virus particles and the cuticule lining the gene(s) required for nematode transmission and en- oesophageal lumen for a nonstructural gene product coded a CP that was able to encapsidate viral RNAs but linking the two. This resulted in the hypothesis that the was nonfunctional in nematode transmission. The DI7 40-kDa protein may play a role similar to the function of RNA transcribed from the nuclear transgene accumu- the helper component (HC-Pro) of potyviruses in the lated at relatively low levels in the healthy transgenic transmission of these viruses by aphids (Herna´ndez et plants but was rapidly amplified upon mechanical inoc- al., 1997). When the leaves of transgenic DI7 plants were ulation of the plants with the homologous TRV isolate inoculated with TRV-PpK20 the DI7 RNA interfered with PpK20 or a serologically different isolate PaY4. The accumulation of wt RNA 2 in the roots of these plants, transgenic DI7 RNA efficiently interfered with the accu- and DI7 was the major RNA-2-specific molecule detect- mulation of wt RNA 2 of TRV-PpK20, and the DI7-encoded able in roots. However, nematodes feeding on these truncated CP (mCP) was the major CP detectable in roots selectively transmitted only virus particles contain- transgenic plants infected with TRV-PpK20. The data ing wt RNA 2. To explain this specificity, we propose that indicated that interference by DI7 with accumulation of wt RNA 2 is encapsidated in cis by its encoded wt CP, wt RNA 2 of isolate PaY4 was slightly less efficient, but whereas DI7 RNA is encapsidated in cis by its encoded this has not been further investigated. mCP. If the nematodes are able to acquire only particles Transgenic expression of DI RNAs derived from to- containing wt CP, this would allow them to exclude DI7 162 VISSER ET AL.

FIG. 7. Accumulation of wt RNA 2 and DI7 RNA in roots of the source and bait plants used in the experiments of Series 2 in Table 1. (A) The accumulation of viral RNAs in the roots of tobacco source plants: nontransgenic plants (lanes 1–6), transgenic D7-6 plants (lanes 7–12) and transgenic D7-7 plants (lanes 13–18). The source plants were inoculated with TRV-PpK20 and nematodes were given access to the root system. After 4 weeks, the nematodes were transferred to the roots of bait plants, and RNA extracted from the roots of the source plants was analyzed by Northen blot hybridization. (B) The accumulation of viral RNAs in the roots of nontransgenic N. clevelandii bait plants infected by virus that was transmitted by nematodes from nontransgenic source plants (lanes 1–6), D7-6 source plants (lanes 7–12) or D7-7 source plants (lanes 13–18). After the nematodes were given an access period of 4 weeks, RNA was extracted from the roots of the bait plants and analyzed by Northern blot hybridization. The blots of (A) and (B) were hybridized with a 32P-labbeled probe corresponding to nucleotides 1–334 of PpK20 RNA 2. Lanes WT and DI7 contain RNA 2 and DI7 RNA as markers, respectively. The positions of RNA 2 and DI7 RNA are indicated in the left margin.

RNA from transmission. Previously we have observed CP and mCP. Possibly, when mixed encapsidation oc- encapsidation in cis for alfalfa mosaic virus (AMV), a curs, a small proportion of wt CP in the capsid may member of the Bromoviridae. The AMV CP is encoded by enable the nematodes to acquire particles containing RNA 3 of the tripartite AMV genome and is translated RNA 1. Because in the sucrose gradient analyzed in Fig. from a subgenomic messenger. Available evidence indi- 8 the particles containing RNA 1, RNA 2, and DI7 RNA cates that this CP encapsidates AMV RNAs 1 and 2 in were not fully separated and the sedimentation pattern trans but RNA 3 in cis (Neeleman and Bol, 1999). Re- may have been affected by aggregation of particles, the cently a functional coupling between replication and mechanism of encapsidation of TRV requires further in- packaging of poliovirus RNA has been reported (Nugent vestigation. However, the observation that mCP and wt et al., 1999). As DI RNAs derived from TRV-PpK20 RNA 2 CP largely cosedimented with DI7 RNA and RNA 2, interfere with the accumulation of wt RNA 2, but not with respectively, is in line with the notion that these RNAs are accumulation of RNA 1, it was suggested that TRV RNAs encapsidated in cis by their encoded CPs. 1 and 2 use separate pools of viral replicase for their Heterologous encapsidation has been observed in amplification. Similarly the replication of TRV RNA 2, the numerous cases of natural mixed infections with viruses synthesis of the subgenomic CP mRNA, and encapsida- belonging to diverse families, particularly among Poty- tion of RNA 2 could be closely integrated in the infected viridae (Nelson and Wheeler, 1981) and Luteoviridae (Hu cell. In transgenic DI7 plants infected with TRV-PpK20, et al., 1988; Wen and Lister, 1991). Isolates defective in RNA 1 may be encapsidated in trans by a mixture of wt vector transmission may become transmissible after het- TRANSMISSION OF TRV BY NEMATODES 163

FIG. 8. Analysis of the encapsidation of viral RNAs in N. benthamiana plants inoculated with a mixture of TRV-PpK20 RNA 1 and infectious cDNAs encoding RNA 2 and DI7 RNA. Progeny virus was centrifuged in a sucrose gradient and viral RNAs and CPs in 13 fractions of the gradient (lanes 1–13) were analyzed by Northern blotting (A) and Western blotting (B), respectively. Sedimentation was from left to right. The Northern blot was hybridized with 32P-labeled probes corresponding to nucleotides 1–334 of RNA 2 and to the 29-kDa movement protein gene in RNA 1. The Western blot was analyzed with an antiserum detecting TRV-PpK20 CP and mCP. Lane 14 of (A) was run with the virus preparation that was loaded on the sucrose gradient; lane 14 of (B) was loaded with virus that did not contain DI7 RNA. The positions of RNA 1, RNA 2, and DI7 RNA are indicated in the left margin of (A). The positions of CP (encoded by RNA 2) and mCP (encoded by DI7 RNA) are indicated in the left margin of (B). erologous encapsidation by CPs of transmissible iso- tobacco, 10 ␮g of either plasmid pCaK20–2T16 or lates (Simons, 1976; Hull, 1977; Bourdin and Lecoq, 1991). pCaTD7 (which were previously linearized with BglI) or Possibly, the high frequency of the loss of genes required 20 ␮g transcript RNA derived from plasmid PaY4-Pt7blue for nematode transmission during replication in the plant were combined with 100 ␮g of total RNA extracted from cell has resulted in tobraviruses developing a strategy to RNA-1-infected tobacco leaves in 300 ␮l of phosphate use vector transmission as a bottleneck to eliminate buffer (18 mM Na2HPO4, pH 7.0). These mixtures were defective quasispecies from the virus population. These used to inoculate 6- to 8-week-old N. tabacum (cv Sam- quasispecies could include DI RNAs emerging due to sun NN) plants (25 ␮l/half-leaf). To produce larger their increased fitness for replication in the host plant. amounts of inoculum, the primary infection was pas- Pirone and Blanc (1996) first discussed this “bottleneck” saged by mechanical sap inoculation to new plants. principle with regard to transmission. These However, to avoid the emergence of natural defective authors consider vector transmission to have a large interfering RNAs, this procedure was carried out only impact on the dynamics of virus populations and dis- once. Infection of N. benthamiana was done with an cussed how viruses may cope with two, apparently con- inoculum that contained 100 ␮g RNA from RNA 1 in- flicting, adaptive problems, namely fitness in the host fected plants, 10 ␮g of plasmid pCaK20–2T16, and 1 ␮g plant and vector transmissibility. Our data indicate that of plasmid pCaTD7 per 300 ␮l of phosphate buffer. Three tobraviruses have developed a unique strategy to apply half leaves per plant were inoculated, and virus was the bottleneck principle. isolated 7 days after inoculation from the inoculated and systemically infected leaves as described (Angenent et MATERIALS AND METHODS al., 1986) using PCA buffer (0.18 M Na2HPO4, 20 mM citric acid, 2 mM ␤-mercaptoethanol, pH 7.0) for homogeniza- Virus inoculation and purification tion of the leaves and storage of the virus. Purified virus (4.5 mg) was loaded on a 6–30% sucrose gradient and TRV isolates PpK20 and PaY4 are described by Ploeg centrifuged for3hat58,000g at 4°C in a Beckman et al. (1992). Infectious cDNA clones of the full-length SW-27.1 rotor. Thirty-five fractions of 0.5 ml were col- RNA2 (plasmid pCaK20–2T16) and the defective interfer- lected, and the composition of 13 fractions that contained ing RNA DI7 (plasmid pCaTD7) of isolate PpK20 were viral material was analyzed by Northern and Western constructed by Herna´ndez et al., (1996). An infectious blotting. Before Northern blot analysis, RNA was ex- cDNA clone of RNA 2 of isolate PaY4 (PaY4-Pt7blue), tracted from the virus particles as described (Angenent which is driven by a T7 promoter, was a gift of S. A. et al., 1986). MacFarlane (SCRI, Scotland, UK). Purified RNA 1 of iso- late PpK20 was used to produce nonmultiplying infec- Transformation of plants tions in tobacco plants and maintained by using total RNA extracts from the primary infected leaf material as Plasmid pCaTD7 was partially digested with PvuII. The inocula. To produce complete “multiplying” infections in fragment containing the complete DI7 sequence flanked 164 VISSER ET AL. by the 35S promoter and nos terminator was ligated into plants, or vice versa, with nontransgenic N. clevelandii the SmaI site of pUC19, resulting in subclone pUC19D7. plants as source plants, and transgenic N. tabacum The fragment was cut from this subclone using the plants as bait plants. The virus transmission experiments unique KpnI and HindIII sites of the pUC19D7 polylinker were further performed by standard procedures (Mac- and was subsequently ligated into the KpnI–HindIII-di- Farlane et al., 1995). All plants were grown individually in gested transformation vector pMOG800. PMOG800 con- 25-cm3 plastic pots. Groups of 60 nematodes were tains the NPTII gene for selection on kanamycin and the placed into each pot, and a source plant that, 2 days left and right border for transfer by A. tumefaciens. This previously, had been mechanically inoculated with virus construct was transformed via A. tumefaciens strain was added. The nematodes were recovered after 4 LBA4404 to N. tabacum cv Samsun NN using the leaf weeks access to the roots of the virus-infected plants disk transformation procedure (Horsch et al., 1985). and transferred to a new pot containing a healthy bait Transgenic tissue was selected with 200 ␮g/ml kanamy- plant. The nematodes were given a further period of 4 cin and 200 ␮g/ml carbenicilline. Primary transformants weeks to transmit the virus to the bait plants. Roots were allowed to self-pollinate, and T1 seed was germi- systems of all source and bait plants were stored at nated on Murashige and Skoog medium containing 200 Ϫ70°C or directly used for further analysis. Half of each ␮g/ml kanamycin, after which surviving plantlets were root system was comminuted with a mortar and pestle transferred to soil. and the resultant suspension rubbed by finger onto Car- borundum-dusted leaves of Chenopodium quinoa and C. RNA and protein analysis amaranticolor virus indicator plants. From the other half of the root system, RNA was recovered and submitted to Total RNA from inoculated leaves was isolated 5 days Northern blotting to examine the identity of the transmit- p.i. by phenol extraction and lithium chloride precipita- ted virus. tion (Verwoerd et al., 1989). Two micrograms of RNA was glyoxalated and electrophoresed in a 1.5% agarose gel using 15 mM phosphate buffer. Viral RNAs were visual- ACKNOWLEDGMENTS ized by Northern blot hybridization as described by Her- na´ndez et al., (1996). RNA 2 of TRV isolate PpK20 and DI7 The assistence of Mrs. Sheena Lamond with the vector transmission experiments is gratefully acknowledged. P.B.V. is financially supported RNA was detected on the blots by using probes corre- by the Association of Biotechnical Centres in The Netherlands (ABON). sponding to nucleotides 1–151 or 1–334 of RNA 2. RNA 1 D.J.F.B. and J.F.B. gratefully acknowledge financial assistence received was detected by using a probe corresponding to the under the NWO-British Council Joint Scientific Research Projects full-length open reading frame of the 29-kDa movement scheme. Research at the Scottish Crop Research Institute is grant- protein gene. To detect RNA 2 of TRV isolate PaY4, a aided by the Scottish Office of Agriculture, Environment and Fisheries Department (SOAEFD). Experiments with transgenic plants were done probe was used corresponding to nucleotides 84–540 of under license from SOAEFD. TRV-PaY4 RNA 2. 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