Institute of Molecular Plant Sciences, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands1
Author for correspondence: John F. Bol. Fax +31 71 5274340. e-mail j.bol{at}chem.leidenuniv.nl
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nematode transmission studies with pseudorecombinant TRV isolates produced from the nontransmissible isolate PLB and the transmissible isolate PpK20 showed that vector transmissibility segregated with RNA 2 (Ploeg et al., 1993 ). A mutational analysis of genes in RNA 2 of isolates TRV-PpK20 and PEBV-TpA56 showed that CP and nonstructural proteins are both involved in the transmission process. In addition to the 5' proximal CP gene (i.e. the 2a gene), TRV-PpK20 RNA 2 contains the 2b and 2c genes encoding a 40 kDa protein (40K) (formerly designated as 29·4K) and a 32·8 kDa protein (32·8K), respectively (Hernández et al., 1995
; Visser et al., 1999b
). Deletions in the 40K gene interfered with transmission of TRV-PpK20 by its vector nematode Paratrichodorus pachydermus, whereas deletions in the 32·8K gene did not (Hernández et al., 1997
). It was suggested that the 32·8K gene could be involved in transmission of TRV-PpK20 by other vector nematode species. In RNA 2 of PEBV-TpA56, the CP gene is followed by reading frames encoding 9K, 29K and 23K proteins and mutation of each of these reading frames affected transmission of the virus by the vector nematode Trichodorus primitivus (MacFarlane et al., 1996
; Schmitt et al., 1998
).
An intriguing aspect of the transmission process is the high specificity in virusvector associations. This concept of specificity is defined with the terms exclusivity, in which there is an apparent unique association between a virus isolate and a vector species, and complementarity, in which different virus isolates may share the same vector species, or conversely, one particular virus isolate may be transmitted by several nematode species (Vassilakos et al., 1997 ; Brown et al., 1995
; Brown & Weischer, 1998
). A correlation was found between the serotype of a particular Tobravirus isolate and its vector nematode specificity, suggesting that features on the virus particle determine specific recognition by the vector (Ploeg et al., 1992
). Studies on the particle structure of tobraviruses by nuclear magnetic resonance spectroscopy revealed that a protruding, mobile segment was located at the C terminus of the CP subunit (Mayo et al., 1993
). Partial deletion of this mobile segment interfered with transmission of PEBV by its vector nematode (MacFarlane et al., 1996
).
The requirement for helper protein(s) in vector transmission is not uncommon for plant viruses that are transmitted in a noncirculative manner. The role and function in aphid transmission of HC-Pro of potyviruses and ATF of caulimoviruses has been thoroughly studied (for reviews see Hull, 1994 ; Schmidt et al., 1994
; Gray, 1996
; Pirone & Blanc, 1996
). Present knowledge about the function of these helper proteins of poty- and caulimoviruses supports the bridge hypothesis initially proposed by Govier & Kassanis (1974)
. The helper proteins would contain two distinct functional domains, one interacting with the virus particle and the other with a specific receptor site in the aphid stylet or foregut. As suggested by Hernández et al. (1997)
, similarities between the mode of tobravirus transmission by nematodes with insect vectors of poty- and caulimoviruses may reflect a common molecular mechanism of virus transmission. The 40K protein of TRV-PpK20 and the RNA 2-encoded nonstructural proteins of PEBV-TpA56 could act as helper proteins with functions in nematode transmission similar to that of HC-Pro and ATF in aphid transmission of poty- and caulimoviruses.. So far, possible interactions between CP and putative tobravirus helper components, or between helper components and nematodes, have not been investigated.
Here, we showed that the TRV-PpK20 40K protein is expressed in leaves and roots of infected plants and that this expression is correlated with the expression of the CP gene. By using the yeast two-hybrid system, we observed that CP of TRV-PpK20 interacts with the 40K and 32·8K proteins and that the C terminus of CP is specifically involved in these interactions.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Preparation of antisera.
Bands containing 32·8K or 40K proteins were excised from Coomassie-stained polyacrylamide gels, homogenized in Freunds incomplete adjuvant and used for the immunization of rabbits (Eurogentec). Injections were subcutaneous, the first with 100 µg of purified proteins (day 0), followed by three boosts, each with 50 µg of proteins at intervals of 14, 14 and 18 days. Blood samples were taken at days 0 (pre-immune serum), 38, 66 and 80 (final bleeding). Antibody titres of all samples were determined by ELISA (Sambrook et al., 1989). For use in Western blot analysis, sera with the highest titres were partially purified by cross-absorption with acetone-precipitated proteins from healthy plants, as described by Hay et al. (1994) .
Immunodetection of viral proteins in plants.
Total protein extracts from roots or leaves of Nicotiana benthamiana plants infected with TRV-PpK20 were prepared as described by Hernández et al. (1996) . To obtain subcellular protein fractions, 1·0 g of leaf or root material was collected 6 days after inoculation and homogenized with a mortar and pestle at 4 °C in 3·0 ml of homogenization buffer (100 mM TrisHCl, pH 8·0, 10 mM KCl, 5 mM MgCl2, 10% glycerol, 10 mM
-mercaptoethanol) and centrifuged at 1000 g for 15 min. The supernatant was centrifuged at 30000 g for 30 min at 4 °C (Angenent et al., 1989
). Proteins in the 1000 g pellet, 30000 g pellet and 30000 g supernatant were solubilized in Laemmli buffer. For the detection of CP and 40K protein by Western blot analysis (Towbin et al., 1979
), gels were loaded with amounts of the subcellular fractions corresponding to 0·5 and 2·0 mg of fresh leaf material, respectively. The blots were analysed with partially purified antisera against CP of TRV-PLB (Angenent et al., 1989
) and 40K protein of TRV-PpK20 (this work).
Yeast two-hybrid experiments.
Yeast strain pJ69-4A (containing the HIS3 and ADE2 reporter genes; James et al., 1996 ) was transformed with a pACT-II-derived construct, a pAS2-1-derived construct, or with a mixture of a pACT-II- and a pAS2-1-derived construct, as described by Gietz et al. (1995)
. pACT-II and pAS2-1 contain Leu and Trp selection markers, respectively. Single transformants were identified by plating on 2% sucrose-containing SC media, lacking leucine (SC-Leu) for pACT-II-derived constructs or lacking tryptophan (SC-Trp) for pAS2-1-derived constructs. Double transformants were selected on SC-Leu-Trp double selective medium.
To identify proteinprotein interactions resulting in the expression of the HIS3 and ADE2 reporter genes (Fields & Sternglanz, 1994 ), four yeast colonies of the transformants listed in Table 2
were plated on media lacking leucine, tryptophan, histidine and adenine (SC-Leu-Trp-His-Ade) and evaluated for their ability to grow at 30 °C. Colonies of double transformants were considered positive when at least three out of four colonies showed significant growth within 5 days after plating on SC-Leu-Trp-His-Ade medium. Positive transformants were plated on SC-Leu-Trp-His-Ade medium containing 10 mM 3-amino-1.2.4-triazole (3-AT, Sigma) to suppress leaky expression of the HIS3 gene (Durfee et al., 1993
).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Subcellular localization of CP and 40K protein
To investigate the subcellular localization of CP and 40K protein, virus-infected and non-infected leaf and root samples were homogenized and the homogenate was fractionated into a 1000 g pellet, a 30000 g pellet and a 30000 g supernatant. The 1000 g pellet contains nuclei and chloroplasts, the 30000 g pellet contains membrane fractions whereas cytosolic proteins are present in the 30000 g supernatant (Angenent et al., 1989 ). Fig. 4
shows a Western blot analysis of the presence of 40K protein (Fig. 4a)
and CP (Fig. 4 b
) in the three subcellular fractions from roots (lanes 2, 3 and 4) and leaves (lanes 5, 6 and 7). The distribution of the 40K protein over the 1000 g pellet, 30000 g pellet and 30000 g supernatant of root and leaf homogenates closely resembled that of CP. This could point to a possible co-localization of 40K protein with (aggregates of) virus particles. Interestingly, the antiserum against the 40K protein specifically detected a 22K protein in the 30000 g supernatant from the roots of infected plants (Fig. 4a
, lane 4). As this protein is equally present in the same fraction from the roots of healthy plants (Fig. 4c
, lane 4) it is apparently a host protein.
|
No homodimer 32·8K32·8K or 40K40K protein interactions could be observed in the yeast two-hybrid system (Table 2). Also, no heterodimer 32·8K40K protein interaction was detectable, irrespective of whether the proteins were fused to the activation domain or DNA binding domain of GAL4. However, the two-hybrid system did reveal a specific CPCP interaction and interactions between CP and the 32·8K and 40K proteins (Fig. 6
and Table 2
). When the selection pressure was increased by addition of 10 mM 3-AT to the medium, yeast colonies with the CPCP interaction were still obtained but no CP32·8K or CP40K protein interactions were observed (not shown). This may indicate that CPCP interactions observed with the two-hybrid system are stronger than CP32·8K or CP40K protein interactions. In the absence of 3-AT, visible growth of yeast colonies with the CPCP or CP32·8K protein combinations was observed after 3 days whereas growth of colonies with the CP40K protein combination was observed after 4 days. This suggests that the CP40K protein interaction is weaker than the CP32·8K protein interaction.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antisera against the PEBV-TpA56 29K and 23K proteins have been used to demonstrate the expression of the 2b and 2c genes of this isolate in leaves and roots of N. benthamiana plants (Schmitt et al., 1998 ). Similar to the results with the PEBV-TpA56 29K protein, we observed that expression of the TRV-PpK20 40K protein in leaves follows that of CP with a slight delay in the start of 40K protein synthesis and a slight advance in the cessation of expression or protein turnover. Another similarity is the observation that CP and 2b gene products of TRV-PpK20 and PEBV-TpA56 accumulated at variable ratios in the roots of infected plants. The reason for this variation is unclear. The distribution of CP over the 1000 g pellet, 30000 g pellet and 30000 g supernatant from extracts of TRV-PpK20-infected N. benthamiana plants (Fig. 4 b)
was very similar to that observed previously for corresponding subcellular fractions of homogenized tobacco protoplasts (Angenent et al., 1989
). In this previous study, the 16K nonstructural protein encoded by TRV-PpK20 RNA 1 was found to be exclusively present in the 30000 g pellet. In contrast, the distribution of the 40K protein over the three subcellular fractions closely resembled that of CP (Fig. 4a)
. If the 40K protein is physically attached to virus particles, the interaction is apparently disrupted by the virus isolation procedure as no 40K protein was detectable by Western blot analysis in purified virus preparations (results not shown).
In addition to the co-localization of CP and 40K protein in subcellular fractions, an interaction was observed between the two proteins in the yeast two-hybrid system. Although the interaction was weaker than the interaction observed between CP subunits, it was apparently specific. Deletion of the C-terminal 19 aa of CP abolished the interaction with the 40K protein but not the interaction with the full-length CP. From comparisons of tobamoviral CPs, it has been predicted that internal domains mediate interactions between CP subunits in virions (Goulden et al., 1992 ). Replacement of the C-terminal 15 aa of CP of TRV-PpK20 by three nonviral aa did not affect virion formation (Hernández et al., 1996
). The C terminus of tobraviral CP is predicted to extend away from the virion surface as a flexible arm with a length of 22 aa in TRV-PpK20, 17 aa in TRV-TpO1 and 29 aa in PEBV-TpA56 (Mayo et al., 1993
; MacFarlane et al., 1999
). Removal of the C-terminal 15 aa of CP of PEBV-TpA56 (but retaining the terminal alanine residue) abolished nematode transmission of the virus (MacFarlane et al., 1996
). Our observation that deletion of the C-terminal 19 aa of CP of TRV-PpK20 interfered with its interaction with the 40K protein in the two-hybrid system indicates that a CP40K protein interaction plays a role in the transmission process. This observation is in line with the hypothesis that the 40K protein forms a bridge between putative receptors in the food canal of the vector nematode and specific domains of CP on the surface of virus particles (Brown et al., 1995
; Hernández et al., 1997
). However, we cannot rule out the possibility that the nonviral amino acids at the C terminus of CP
19 affected the interaction of this protein with the 40K protein. The sequence of the C-terminal 15 aa of CP of PEBV-TpA56 and TRV-TpO1 is almost identical but very different from the C-terminal sequence of CP of TRV-PpK20. It would be interesting to see whether a possible specificity in CP2b protein interactions would correlate with vector specificity of these three viruses.
The 32·8K protein encoded by the TRV-PpK20 2c gene is neither required for transmission by P. pachydermus nor for replication of mechanically inoculated virus, and it has been speculated that this 32·8K protein may play a role in transmission of TRV-PpK20 by vector nematodes other than P. pachydermus (Hernández et al., 1997 ). Because preparation of an antiserum against this protein was not successful, we could not investigate a possible common subcellular localization of CP and 32·8K protein. However, similar to the 40K protein, the 32·8K protein did interact with CP in the two-hybrid system. A CP deletion analysis pointed to a determinant involved in this interaction that is located between aa 19 and 79 from the C terminus.
A direct interaction between the CP and HC-Pro of potyviruses has been demonstrated by using a protein blotting-overlay technique (Blanc et al., 1997 ). In that study, HC-Pro was purified from infected plants as a biologically active dimer (Thornbury et al., 1985
). By the use of the two-hybrid system, PVA CPCP and HC-ProHC-Pro dimer formation could be demonstrated but no CPHC-Pro interaction was detectable (Guo et al., 1999
). We did not detect dimer formation of the 40K or 32·8K proteins in the two-hybrid system, indicating that the 40K protein functions as a monomer in transmission of TRV-PpK20 by the vector nematode P. pachydermus.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blanc, S., López-Moya, J.-J., Wang, R., García-Lampasona, S., Thornbury, D. W. & Pirone, T. P. (1997). A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Virology 231, 141-147.[Medline]
Brown, D. J. F. & Weischer, B. (1998). Specificity, exclusivity and complementarity in the transmission of plant viruses by plant parasitic nematodes: an annotated terminology. Fundamental and Applied Nematology 21, 1-11.
Brown, D. J. F., Robertson, W. M. & Trudgill, D. L. (1995). Transmission of viruses by plant nematodes. Annual Review of Phytopathology 33, 223-249.
Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Lee, W.-H. & Ellegge, S. J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes & Development 7, 555-569.[Abstract]
Fields, S. & Sternglanz, R. (1994). The two-hybrid system: an assay for proteinprotein interactions. Trends in Genetics 10, 286-292.[Medline]
Frangioni, J. V. & Neel, B. G. (1993). Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Analytical Biochemistry 210, 179-187.[Medline]
Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. (1995). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355-360.[Medline]
Goulden, M. G., Davies, J. W., Wood, K. R. & Lomonossof, G. P. (1992). Structure of tobraviral particles: a model suggested from sequence conservation in tobraviral and tobamoviral coat proteins. Journal of Molecular Biology 227, 1-8.[Medline]
Govier, D. A. & Kassanis, B. (1974). A virus-induced component of plant sap needed when aphids acquire potato virus Y from purified preparations. Virology 61, 420-426.[Medline]
Gray, S. M. (1996). Plant virus proteins involved in natural vector transmission. Trends in Microbiology 4, 259-264.[Medline]
Guo, D., Merits, A. & Saarma, M. (1999). Self-association and mapping of interaction domains of helper component-proteinase of potato A potyvirus. Journal of General Virology 80, 1127-1131.[Abstract]
Harrison, B. D. & Robinson, D. J. (1986). Tobraviruses. In The Plant Viruses, pp. 339-369. Edited by H. V. Van Regenmortel & H. Fraenkel-Conrat. New York: Plenum Press.
Hay, J., Grieco, F., Druka, A., Pinner, M., Lee, S.-C. & Hull, R. (1994). Detection of rice tungro bacilliform virus gene products in vivo. Virology 205, 430-437.[Medline]
Hernández, C., Mathis, A., Brown, D. J. F. & Bol, J. F. (1995). Sequence of RNA 2 of a nematode-transmissible isolate of tobacco rattle virus. Journal of General Virology 76, 2847-2851.[Abstract]
Hernández, C., Carette, J. E., Brown, D. J. F. & Bol, J. F. (1996). Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA2. Journal of Virology 70, 4933-4940.[Abstract]
Hernández, C., Visser, P. B., Brown, D. J. F. & Bol, J., F. (1997). Transmission of tobacco rattle virus isolate PpK20 by its nematode vector requires one of the two non-structural genes in the viral RNA 2. Journal of General Virology 78, 465467.[Abstract]
Hull, R. (1994). Molecular biology of plant virusvector interactions. In Advances in Disease Vector Research, pp. 361-386. Edited by K. F. Harris. New York: Springer-Verlag.
James, P., Halladay, J. & Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425-1436.
MacFarlane, S. A. & Brown, D. J. F. (1995). Sequence comparison of RNA2 of nematode-transmissible and nematode non-transmissible isolates of pea early-browning virus suggests that the gene encoding the 29 kDa protein may be involved in nematode transmission. Journal of General Virology 76, 1299-1304.[Abstract]
MacFarlane, S. A., Wallis, C. V. & Brown, D. J. F. (1996). Multiple virus genes involved in the nematode transmission of pea early browning virus. Virology 219, 417-422.[Medline]
MacFarlane, S. A., Vassilakos, N. & Brown, D. J. F. (1999). Similarities in the genome organization of tobacco rattle virus and pea early browning virus isolates that are transmitted by the same vector nematode. Journal of General Virology 80, 273-276.[Abstract]
Mayo, M. A., Brierley, K. M. & Goodman, B. A. (1993). Developments in the understanding of the particle structure of tobraviruses. Biochimie 75, 639-644.[Medline]
Pirone, T. P. & Blanc, S. (1996). Helper-dependent vector transmission of plant viruses. Annual Review of Phytopathology 34, 227-247.
Ploeg, A. T., Brown, D. J. F. & Robinson, D. J. (1992). The association between species of Trichodorus and Paratrichodorus vector nematodes and serotypes of tobacco rattle tobravirus. Annals of Applied Biology 121, 619-630.
Ploeg, A. T., Robinson, D. J. & Brown, D. J. F. (1993). RNA-2 of tobacco rattle virus encodes the determinants of transmissibility by trichodorid vector nematodes. Journal of General Virology 74, 1463-1466.[Abstract]
Schmidt, I., Blanc, S., Espérandieu, P., Kuhl, G., Devauchelle, G., Louis, C. & Cérutti, M. (1994). Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission. Proceedings of the National Academy of Sciences, USA 91, 8885-8889.[Abstract]
Schmitt, C., Mueller, A., Mooney, A., Brown, D. & MacFarlane, S. A. (1998). Immunological detection and mutational analysis of the RNA2-encoded nematode transmission proteins of pea early browning virus. Journal of General Virology 79, 1281-1288.[Abstract]
Thornbury, D. W., Hellman, G. M., Rhoads, R. E. & Pirone, T. P. (1985). Purification and characterization of potyvirus helper component. Virology 144, 260-267.
Towbin, H. T., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350-4354.[Abstract]
Vassilakos, N., MacFarlane, S. A., Weischer, B. & Brown, D. J. F. (1997). Exclusivity and complementarity in the association between nepo- and tobraviruses and their respective vector nematodes. Mededelingen van de Faculteit Landbouwwetenschappen Universiteit Gent 62, 713-720.
Visser, P. B., Matthis, A. & Linthorst, H. J. M. (1999a). Tobraviruses. In Encyclopedia of Virology, 2nd edn. Edited by A. Granoff & R. G. Webster. London: Academic Press (in press).
Visser, P. B., Brown, D. J. F. & Bol, J. F. (1999b). Nematode transmission of tobacco rattle virus serves as a bottleneck to clear the virus population from defective interfering RNAs. Virology (in press).
Received 17 June 1999;
accepted 13 September 1999.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |