Virology 300, 118 124 (2002) doi:10.1006/viro.2002.1511 Immunogold Localization of Tobravirus 2b Nematode Transmission Helper Protein Associated with Virus Particles Evangelos Vellios, George Duncan, Derek Brown, and Stuart MacFarlane 1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom Received February 13, 2002; returned to author for revision April 4, 2002; accepted April 4, 2002 Transmission of the tobraviruses Tobacco rattle virus (TRV) and Pea early-browning virus (PEBV) by trichodorid vector nematodes requires the viral coat protein (CP) and the 2b protein, a nonstructural protein encoded by RNA2, the smaller of the two viral genomic RNAs. It is hypothesized that the 2b protein functions by interacting with a small, flexible domain located at the C-terminus of the CP, forming a bridge between the virus particle and the internal surface of the vector nematode feeding apparatus. Antibodies specific for the 2b protein of PEBV or TRV did not bind to virus particles that were adsorbed to electron microscope grids and were not able to trap virus particles from extracts of infected plants. However, electron microscopy of thin sections of plants infected with PEBV probed with 2b-specific antibodies which were further gold-labeled showed that the 2b protein localizes exclusively to virus particles. Similarly, immunogold localization studies showed that the 2b protein of TRV isolate PaY4 is associated only with TRV PaY4 virus particles. When a recombinant TRV encoding the PaY4 2b protein and the CP from TRV isolate PpK20 was examined, the 2b protein could not be detected by Western blotting and in IGL experiments was not associated with virus particles. These results suggest that in the absence of a specific interaction between compatible CP and 2b proteins, the 2b protein does not accumulate. 2002 Elsevier Science (USA) INTRODUCTION The three tobraviruses Tobacco rattle virus (TRV), Pea early-browning virus (PEBV), and Pepper ringspot virus (PepRSV)are transmitted between plants by trichodorid nematodes belonging to the genera Trichodorus and Paratrichodorus. Studies with pseudorecombinant and mutated viruses have shown that the viral determinants for nematode transmission are located on RNA2, the smaller of the two viral genomic RNAs (Ploeg et al., 1993; MacFarlane et al., 1996; Hernandez et al., 1997). Sequencing of RNA2 from more than 12 tobravirus isolates has revealed extensive diversity in both primary nucleotide sequence and gene content (MacFarlane, 1999). Commonly this variation results from recombination between nonhomologous regions of viral RNAs 1 and 2, leading to the replacement of 3 -terminal sequences of RNA2 with those of RNA1. As a result of this process, in laboratory strains, RNA2-specific genes frequently are deleted. Infectious clones have been constructed for four nematode-transmissible tobravirus isolates. RNA2 of the TpA56 isolate of PEBV, which is transmitted by Trichodorus primitivus, encodes four proteins, the coat protein (CP), a 9-kDa protein, a 29-kDa 2b protein, and a 23-kDa 2c protein. The CP, 2b, and 2c proteins have been detected by Western 1 To whom correspondence and reprint requests should be addressed. Fax: 44 1382 562426. E-mail: s.macfarlane@scri.sari.ac.uk. blotting, whereas the 9K protein was not detected by Western blotting and, thus, may not be expressed (MacFarlane and Brown, 1995; Schmitt et al., 1998). TRV isolate TpO1, which also is transmitted by T. primitivus, encodes a CP, 9-kDa, 29-kDa 2b, and 18-kDa 2c proteins. TRV PpK20, which is transmitted by Paratrichodoros pachydermus, encodes a CP, 40-kDa 2b, and 33-kDa 2c proteins (Hernandez et al., 1995; MacFarlane et al., 1999). TRV PaY4, which is transmitted by both P. pachydermus and P. anemones, encodes CP, 27-kDa 2b, and 32-kDa 2c proteins (Vassilakos et al., 2001). There is some, albeit limited, amino acid sequence homology between the 2b proteins from different tobravirus isolates, with the 2b proteins of PEBV TpA56 and TRV TpO1 being the most closely related of the four isolates described above. Possibly this reflects the fact that both viruses are transmitted by the same vector nematode. Conversely, the 2b proteins of TRV PaY4 and PpK20 are not closely related, even though they share a common vector nematode. The 2c proteins of these four isolates have no significant sequence similarities and are classed together only by their similar location in the viral genome. Mutagenesis experiments showed that for PEBV TpA56 the 2b protein was essential for nematode transmission (MacFarlane et al., 1996). Similarly the 2b proteins of both TRV PpK20 and PaY4 are essential for transmission of these viruses (Hernandez et al., 1997; Vassilakos et al., 2001). For PEBV Tp A56, mutation of the 2c gene greatly reduced but did not completely abolish transmission. This contrasts with TRV PpK20 and PaY4, 0042-6822/02 $35.00 2002 Elsevier Science (USA) All rights reserved. 118
IMMUNOGOLD LOCALIZATION OF TOBRAVIRUS 2b PROTEIN 119 where mutation of the 2c gene had no effect on the frequency of transmission. Again, this may reflect the fact that PEBV TpA56 is transmitted by a different nematode species to TRV PpK20 and PaY4. The viruses are transmitted in a noncirculative manner, which means that virus particles are taken up into the nematode pharyngeal tract during feeding where they are retained by a specific attachment. The virus particles are released, perhaps by a ph change in the pharyngeal tract during subsequent feeding of the nematode on another plant (Taylor and Brown, 1997). Immunogold localization (IGL)studies of nematodes that have fed on plants infected with different tobravirus isolates showed that virus particles are closely associated with the cuticle lining the lumen of the posterior part of the pharyngeal bulb (Karanastasi et al., 2000a,b). The specificity of this binding interaction was highlighted by the fact that in these nematodes virus particles did not attach to other parts of the pharyngeal tract such as the anterior region or to the pharyngostome. Nematodes that fed on plants infected with a tobravirus isolate that could not be transmitted by the nematode did not retain virus particles in any regions of the pharyngeal tract (Karanastasi, 2001). Thus, the retention of virus within the nematode pharyngeal tract is likely to be the step that determines whether transmission is successful. Several approaches have been used to analyze the structure of TRV particles, including comparative alignment of the amino acid sequence, NMR, epitope mapping, and Raman optical activity (Goulden et al., 1992; Brierley et al., 1993; Legorburu et al., 1996; Blanch et al., 2001). One conclusion from these studies is that the C-terminal region of the TRV CP is fully exposed on the surface of the particle and is, thus, accessible for interaction with other viral or nematode proteins. The importance of this region was demonstrated by mutation analysis. For PEBV TpA56, deletion either of the complete 28 amino acid C-terminal domain or only of the 15 C-terminal residues abolished nematode transmission of the virus (MacFarlane et al., 1996). Similarly, for TRV PpK20, replacement of 15 amino acids at the C-terminus of the CP with three nonviral residues prevented nematode transmission (Hernandez et al., 1996). Interactions between the TRV CP and nonstructural 2b and 2c proteins were examined using the yeast two-hybrid system (Visser and Bol, 1999). This study found that the CP interacted with itself, with the 2b protein, and with the 2c protein. Interestingly, deletion of 19 amino acids at the C-terminus of the CP prevented its interaction with the 2b protein but did not affect interaction with itself or the 2c protein. These findings, when taken together with the data showing that the 2b protein is essential for transmission of TRV and PEBV, suggest that interaction between the 2b protein and the C-terminus of the CP is a critical step in the transmission process. Possibly, the 2b protein functions by forming a structural bridge between the tobravirus particle and a specific recognition site(s) on the pharyngeal cuticle of the vector nematode. Results obtained using the yeast two-hybrid system are often criticized as being artifactual. We decided to corroborate the possible interaction between the 2b protein and CP using IGL to visualize the viral proteins in infected plants. RESULTS AND DISCUSSION These studies concentrated primarily on two tobravirus isolates, PEBV TpA56 and TRV PaY4, for which nematode-transmissible, infectious cdna clones of RNA2 and antisera specific for each of the virus CP and 2b proteins were available. A variety of tests were carried out to determine whether the 2b nematode-transmission helper protein was physically associated with virus particles. Immunosorbent electron microscopy (ISEM) and antibody coating tests ISEM relies on the physical trapping of virus particles to a microscope grid by specific antibodies. For this to be achieved the protein that is recognized by the antibodies must maintain its association with virus particle during the procedure and be present in high enough amounts to bring about efficient trapping. Both the PEBV TpA56 and the TRV PaY4 virus-specific antisera were able to trap homologous virus particles (data not shown). In contrast, neither the PEBV TpA56 2b nor the TRV PaY4 2b antisera were able to trap any virus particles in these studies. An attempt to stabilize the virions by extraction of infected leaf material with buffer containing 1% glutaraldehyde also did not bring about trapping of particles by either of the 2b-specific antisera. The antibody-coating experiments produced similar results, with virus-specific antibodies binding to virus particles that were adsorbed onto grids but without obvious binding of 2b-specific antibodies (data not shown). Taken together these results suggest that if, as was shown by yeast two-hybrid experiments, the 2b protein does interact with the CP, the interaction is not strong enough to survive the extraction procedures and/or is not sufficiently extensive to be detected by these techniques. Immunogold localization of 2b protein to PEBV particles Nicotiana benthamiana plants were inoculated with PEBV SP5 RNA1 and transcript of PEBV TpA56 RNA2, and systemic-infected leaf samples were collected at 6 days postinoculation. The leaf samples were processed for IGL and probed initially with antibodies specific for the virus CP. Examination of these tissue sections revealed that PEBV particles accumulated in several types
120 VELLIOS ET AL. FIG. 1. Electron micrographs of sections of PEBV-infected leaf. (A F)Plant infected with wild-type PEBV. (G and H)Plant infected with PEBV mutant 29. (A D and G)Probed with CP-specific antiserum. (E, F, and H)Probed with 2b-specific antiserum. Bar in (A)indicates 1 m; all other bars indicate 100 nm. of aggregation, all of which were labeled with gold particles. Sheets of virus particles were commonly seen. The sheets could extend up to 6.3 m in length (Fig. 1A) and typically were composed of one layer of virus particles aligned side-by-side (Fig. 1B). The regular packing of the virus particles is clearly visible when such a sheet is examined in cross section (Fig. 1C). More loosely packed clumps of virus particles were also observed and were labeled by gold particles when probed with CPspecific antibodies (Fig. 1D). Numerous particles were also seen apparently lying free in the cytoplasm. When leaf sections were probed with antibody specific to the PEBV 2b protein, gold particles were found associated with the aggregated virus particles (Figs. 1E and 1F), with very limited labeling occurring in the cytoplasm or organelles. This is the first direct evidence to show that in plants the tobravirus 2b nematode-transmission helper protein associates physically with the virus particle. As a control, sections were examined from a plant that was infected with PEBV mutant 29 that lacks the 2b gene. In this case, virus particles were clearly labeled with gold when probed with CP-specific antibody (Fig. 1G); however, the particles were not labeled when probed with 2b-specific antibodies (Fig. 1H). Incompatibility between CP and 2b proteins from different TRV isolates Many more isolates of TRV than PEBV have been described, and for TRV there is good evidence that there is specificity in the interaction between the virus isolate and vector nematode species (Ploeg et al., 1992; Taylor and Brown, 1997). Previously we examined the transmission of TRV PpK20, which has P. pachydermus as a vector nematode, and TRV PaY4, which has both P. pachydermus and P. anemones as vector nematodes. It was found that a 2b mutant of TRV PaY4 was transmitted by P. pachydermus when coinoculated to plants with wild-type TRV PaY4 but that the TRV PaY4 2b mutant was not transmitted when coinoculated with wild-type TRV PpK20, even though this isolate also has P. pachydermus as a vector (Vassilakos et al., 2001). This raised the possibility that the TRV PaY4 CP and TRV PpK20 2b proteins might not be compatible with one another for
IMMUNOGOLD LOCALIZATION OF TOBRAVIRUS 2b PROTEIN 121 nematode transmission. To investigate this situation further, two recombinant TRV RNA2 clones were constructed. Clone pk202b-gfp encodes the PpK20 CP, the PpK20 2b protein, and GFP, whereas, clone ppay42b-gfp encodes the PpK20 CP, the PaY4 2b protein, and GFP (Fig. 2A). Clone ppay4 is a full-length cdna clone of RNA2 of wild-type TRV PaY4 that encodes the PaY4 CP, PaY4 2b, and PaY4 2c proteins (Vassilakos et al., 2001). Transcripts from these clones were mixed with TRV PpK20 RNA1 and inoculated to N. benthamiana plants. Both the K202b-GFP and the PaY42b-GFP recombinant viruses produced a rapid, efficient systemic infection, as was confirmed by visualization of GFP expression using UV illumination; however, only the wild-type PaY4 and the recombinant virus K202b-GFP were transmitted by nematodes (data not shown). Western blot analysis of systemically infected leaf tissue showed that virus containing the wild-type PaY4 RNA2 expressed easily detectable levels of the PaY4 2b protein (Fig. 2B, Lane 3). However, in systemic leaves infected with the recombinant virus PaY42b-GFP, although the virus CP was expressed at high levels, expression of PaY4 2b protein could not be detected (Fig. 2B, Lane 2). Although the 2b gene in clone PaY42b-GFP has been modified to include a novel, NcoI-restriction site at the translation initiation codon, this procedure does not alter the amino acid sequence of the 2b protein and would not affect its recognition by the PaY4 2b-specific antiserum. In these blots the TRV PaY4 CP expressed by wild-type PaY4 is not recognized by the TRV PpK20 CP-specific antiserum (Fig. 2B, Lane 3). Likewise, the TRV PpK20 2b protein expressed by recombinant K202b-GFP is not recognized by the PaY4 2b-specific antiserum (Fig. 2B, Lane 1). However, it is certain that the recombinant virus K202b-GFP does express the 2b protein, as this virus is nematode transmissible. Thus, the arrangement of the CP, 2b, and GFP genes in both of these constructs does not prevent 2b expression. Therefore, it is likely that in the recombinant virus PaY42b-GFP the 2b protein also is expressed but that it does not accumulate. FIG. 2. Expression of RNA2-encoded proteins by recombinant TRV. (A)TRV RNA2 constructs used in this work. The genes carried on each construct are denoted by boxes. CP, coat protein gene; black box, TRV PpK20 CP; box with the diagonal line fill, TRV PaY4 CP. The PpK20 2b gene has a vertical line fill, and the PaY4 2b gene has a horizontal line fill. The GFP gene has a white fill. The PaY4 2c gene has a dashed-line fill. (B)Western blot of extracts of plants infected with the recombinant viruses described in (A). Lanes 1 to 3 are extracts of systemic infected leaves, inoculated with extracts of transcript-infected plants and sampled at 10 days postinoculation. Lane 1, pk202b-gfp; Lane 2, ppay42b- GFP; Lane 3, ppay4. The upper lanes are probed with antibody specific to the TRV PpK20 CP. The lower lanes are probed with antibody to the TRV PaY4 2b protein. The asterisk denotes a protein present in all samples that reacts with the 2b antiserum. The location of the PaY4 2b protein is marked with an arrow. Immunogold localization of 2b protein to TRV particles Initially, IGL experiments of TRV used plants infected with the wild-type, PaY4 isolate. As was seen with PEBV, TRV was found to accumulate in extended sheets, loose clumps, and as free particles. In all cases the virus particles were gold-labeled when probed with CP-specific antiserum (Figs. 3A 3C). In addition, probing of infected tissue sections with PaY4 2b-specific antiserum resulted in gold labeling of all types of virus particle accumulation (Figs. 3D 3F). The gold particles bound throughout the length of the virus particle, suggesting that the 2b protein interacts uniformly with the virus particle rather than localizing at a particular position. Thus, for both TRV and PEBV, the 2b protein colocalizes with virus particles in infected plants, supporting the hypothesis that the 2b protein could facilitate nematode transmission by acting as a bridge to link the virus particle to surface structures of the nematode pharyngeal tract. When sections of plants infected with recombinant virus PaY42b-GFP, that encodes the TRV PpK20 CP and the TRV PaY4 2b protein, were examined by IGL, although accumulations of virus particles were clearly visible in the cytoplasm, they were not gold-labeled following probing with PaY4 2b-specific antibodies (Figs. 3G and 3H). Significant gold-labeling of the cytoplasm also did not occur, providing further evidence that the PaY4 2b protein does not accumulate in plants infected with this recombinant virus. These findings may explain an observation that was made when the 29 (2b deletion)mutant of PEBV was examined for nematode transmissibility (MacFarlane et al., 1996). This mutation is predicted to produce an in-
122 VELLIOS ET AL. FIG. 3. Electron micrographs of sections of TRV-infected leaf. (A F)Plant infected with wild-type TRV PaY4. (G and H)Plant infected with recombinant TRV PaY42b-GFP. (A C)Probed with CP-specific antiserum. (D H)Probed with 2b-specific antiserum. Bars indicate 100 nm. frame, 38 amino acid deletion within the PEBV 2b protein; however, when extracts of plants infected with this mutant were examined by Western blotting the 2b protein was not detected. Similarly to the TRV 2b protein, one explanation for these findings could be that the PEBV 2b protein is rapidly degraded in the plant cell unless it associates with virus particles. In the case of the PEBV 29 mutant, perhaps the internal deletion removes sequences that interact directly with the virus CP, or alters the tertiary structure of the 2b protein so that it can no longer interact with the CP. We hypothesize that a compatible binding interaction between the tobravirus CP and 2b proteins is necessary to prevent rapid degradation of the 2b protein. Although the TRV PpK20 CP and PpK20 2b protein can interact with one another, and the TRV PaY4 CP and PaY4 2b protein also can interact with one another, a mixture comprising the PpK20 CP and PaY4 2b (or vice versa, we would predict)is incompatible. In this case, the 2b protein is rapidly degraded and nematode transmission of the recombinant virus is prevented. Thus, although the 2b protein is essential for nematode transmission and might be the viral protein that determines which particular species of nematode transmits each different virus isolate, the interaction between the CP and the 2b protein also is critical in this process. Clearly, further detailed study of the CP and 2b interacting domains will be required to better understand the mechanism of tobravirus transmission by nematodes. MATERIALS AND METHODS Immunosorbent electron microscopy ISEM was done essentially as described by Roberts and Harrison (1979). N. benthamiana plants were inoculated with PEBV TpA56 or TRV, and systemically infected
IMMUNOGOLD LOCALIZATION OF TOBRAVIRUS 2b PROTEIN 123 leaves were collected at 6 days post inoculation (d.p.i.). Leaf samples were homogenized using micromortars (Duncan and Roberts, 1981)in 0.08 M citrate buffer, ph 6.5, or 1% buffered glutaraldehyde and centrifuged briefly to pellet insoluble material. Antibody-coated grids were incubated in leaf extracts at 4 C for a period of up to 12 h. Crude rabbit antisera specific for PEBV virions, TRV PaY4 virions, the PEBV TpA56 2b protein (Schmitt et al., 1998), and the TRV PaY4 2b protein (Vassilakos, 2000) were diluted in Sorenson s phosphate buffer, ph 7.0 prior to use. The grids were stained with 2% sodium phosphotungstate and examined using a Jeol 1200EX or Philips CM-10 electron microscope operating at 80 kv. Antibody coating of virus particles Extracts of virus-infected N. benthamiana plants were prepared as for ISEM. Carbon-filmed copper grids were incubated on 10 l drops of extract at room temperature for 30 s to allow virus particles to be adsorbed. The grids were drained briefly and incubated on 10 l drops of antibody diluted in Sorenson s phosphate buffer for 15 min at room temperature. Antisera specific for PEBV and TRV particles was used at a 1:100 dilution; antisera to the PEBV and TRV 2b protein was used at a range of dilutions from 1:32 to 1:1024. Virus particles adsorbed to the grids were visualized by negative staining with sodium phosphotungstate and examination in the electron microscope. Immunogold localization Tissues were prepared using a method provided by Dr. Biao Ding (Ding et al., 1992). Briefly, systemically infected leaf tissue was sampled at between 6 and 10 d.p.i. and 2 5 mm pieces were fixed using a mixture of 2% formaldehyde and 0.5% glutaraldehyde overnight at 4 C. The tissue samples were dehydrated in an ethanol series of increasing concentration (25 to 100%). Subsequently, the tissue was infiltrated with LR White resin (London Resin Co., U.K.)that was polymerized at 55 C for 16 h. Sections were cut using an Ultracut microtome (Reichert-Jung)and glass knives made on a LKB Knife Maker 7801B. The sections (approximately 100 nm thick) were transferred to pyroxylin-filmed nickel grids for IGL. Immunoglobulins were precipitated from rabbit antisera raised against purified PEBV, TRV N5 (Robinson et al., 1987), which is an antiserum that was shown to react with TRV PaY4 CP in Western blot experiments (Vassilakos, 2000), PEBV TpA56 2b protein, and TRV PaY4 2b protein (Vassilakos, 2000). Thereafter IGL was done essentially as described by Roberts (1994). Construction of recombinant TRV Modifications were made to clone ptrv-gfp (MacFarlane and Popovich, 2000)that carries RNA2 from TRV isolate PpK20 in which most of the 2b and all of the 2c gene is removed and replaced by the gene for the green fluorescent protein (GFP). Two cycles of inverse PCR were used to insert an NcoI site at the start of the 2b gene, and a SpeI site upstream of the duplicated CP promoter that is used to express GFP. Subsequently, the TRV PpK20 2b gene was amplified to add a 5 NcoI site anda3 SpeI site and inserted between the TRV PpK20 CP and GFP genes to produce clone pk202b-gfp (Fig. 2A). Also, the TRV PaY4 2b gene was amplified to add a 5 NcoI site and a 3 SpeI site and inserted between the TRV PpK20 CP and GFP genes to produce clone ppay42b-gfp. The PpK20 and PaY4 2b genes were completely sequenced to confirm that no unintentional base changes had been introduced by the PCR procedure. In these clones the 2b gene is expressed from the native TRV PpK20 2b promoter without alteration to the position at which 2b subgenomic mrna synthesis initiates or at which translation of the 2b gene begins. Inoculation of plants For PEBV, capped transcripts were prepared using pt72a56, a full-length clone of RNA2 of isolate TpA56, or mutant 29, which has a 114 nucleotide (38 amino acids, in-frame)deletion in the central part of the 2b gene (MacFarlane et al., 1996). The transcripts were mixed with total RNA isolated from plants infected with RNA1 only of PEBV isolate SP5 and inoculated to plants as described before (MacFarlane et al., 1991). For TRV, capped transcripts were prepared from clones pk202b- GFP, ppay42b-gfp, and ppay4, a full-length clone of RNA2 of TRV PaY4 (Vassilakos et al., 2001). The transcripts were mixed with total RNA isolated from plants infected with RNA1 only of TRV PpK20 before inoculation to plants. ACKNOWLEDGMENTS We thank Sheena Lamond for help with transmission experiments. E.K.V. is supported by an EU TMR Grant (FAIR-BM-973730). SCRI is grant-aided by the Scottish Executive Environment and Rural Affairs Department. 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