Abstract
Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.
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Selected References
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- A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
- Abel P. P., Nelson R. S., De B., Hoffmann N., Rogers S. G., Fraley R. T., Beachy R. N. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science. 1986 May 9;232(4751):738–743. doi: 10.1126/science.3457472. [DOI] [PubMed] [Google Scholar]
- Alexander K. A., Wakim B. T., Doyle G. S., Walsh K. A., Storm D. R. Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein. J Biol Chem. 1988 Jun 5;263(16):7544–7549. [PubMed] [Google Scholar]
- Altschuh D., Lesk A. M., Bloomer A. C., Klug A. Correlation of co-ordinated amino acid substitutions with function in viruses related to tobacco mosaic virus. J Mol Biol. 1987 Feb 20;193(4):693–707. doi: 10.1016/0022-2836(87)90352-4. [DOI] [PubMed] [Google Scholar]
- Asselin A., Zaitlin M. Characterization of a second protein associated with virions of tobacco mosaic virus. Virology. 1978 Nov;91(1):173–181. doi: 10.1016/0042-6822(78)90365-3. [DOI] [PubMed] [Google Scholar]
- Butler P. J., Bloomer A. C., Finch J. T. Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH. J Mol Biol. 1992 Mar 20;224(2):381–394. doi: 10.1016/0022-2836(92)91002-7. [DOI] [PubMed] [Google Scholar]
- Clark M. F., Adams A. N. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol. 1977 Mar;34(3):475–483. doi: 10.1099/0022-1317-34-3-475. [DOI] [PubMed] [Google Scholar]
- Clark W. G., Fitchen J. H., Beachy R. N. Studies of coat protein-mediated resistance to TMV. I. The PM2 assembly defective mutant confers resistance to TMV. Virology. 1995 Apr 20;208(2):485–491. doi: 10.1006/viro.1995.1179. [DOI] [PubMed] [Google Scholar]
- Clark W. G., Fitchen J., Nejidat A., Deom C. M., Beachy R. N. Studies of coat protein-mediated resistance to tobacco mosaic virus (TMV). II. Challenge by a mutant with altered virion surface does not overcome resistance conferred by TMV coat protein. J Gen Virol. 1995 Oct;76(Pt 10):2613–2617. doi: 10.1099/0022-1317-76-10-2613. [DOI] [PubMed] [Google Scholar]
- Davies D. R., Padlan E. A., Sheriff S. Antibody-antigen complexes. Annu Rev Biochem. 1990;59:439–473. doi: 10.1146/annurev.bi.59.070190.002255. [DOI] [PubMed] [Google Scholar]
- Ehrhardt M. R., Erijman L., Weber G., Wand A. J. Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex. Biochemistry. 1996 Feb 6;35(5):1599–1605. doi: 10.1021/bi951267r. [DOI] [PubMed] [Google Scholar]
- FRAENKEL-CONRAT H. Degradation of tobacco mosaic virus with acetic acid. Virology. 1957 Aug;4(1):1–4. doi: 10.1016/0042-6822(57)90038-7. [DOI] [PubMed] [Google Scholar]
- Fenczik C. A., Padgett H. S., Holt C. A., Casper S. J., Beachy R. N. Mutational analysis of the movement protein of odontoglossum ringspot virus to identify a host-range determinant. Mol Plant Microbe Interact. 1995 Sep-Oct;8(5):666–673. doi: 10.1094/mpmi-8-0666. [DOI] [PubMed] [Google Scholar]
- Fitchen J. H., Beachy R. N. Genetically engineered protection against viruses in transgenic plants. Annu Rev Microbiol. 1993;47:739–763. doi: 10.1146/annurev.mi.47.100193.003515. [DOI] [PubMed] [Google Scholar]
- Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hackland A. F., Rybicki E. P., Thomson J. A. Coat protein-mediated resistance in transgenic plants. Arch Virol. 1994;139(1-2):1–22. doi: 10.1007/BF01309451. [DOI] [PubMed] [Google Scholar]
- Hemenway C., Fang R. X., Kaniewski W. K., Chua N. H., Tumer N. E. Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA. EMBO J. 1988 May;7(5):1273–1280. doi: 10.1002/j.1460-2075.1988.tb02941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holt C. A., Beachy R. N. In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants. Virology. 1991 Mar;181(1):109–117. doi: 10.1016/0042-6822(91)90475-q. [DOI] [PubMed] [Google Scholar]
- Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
- Lu B., Stubbs G., Culver J. N. Carboxylate interactions involved in the disassembly of tobacco mosaic tobamovirus. Virology. 1996 Nov 1;225(1):11–20. doi: 10.1006/viro.1996.0570. [DOI] [PubMed] [Google Scholar]
- Martin S. R., Bayley P. M., Brown S. E., Porumb T., Zhang M., Ikura M. Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule. Biochemistry. 1996 Mar 19;35(11):3508–3517. doi: 10.1021/bi952522a. [DOI] [PubMed] [Google Scholar]
- Meador W. E., Means A. R., Quiocho F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992 Aug 28;257(5074):1251–1255. doi: 10.1126/science.1519061. [DOI] [PubMed] [Google Scholar]
- Namba K., Pattanayek R., Stubbs G. Visualization of protein-nucleic acid interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9 A resolution by X-ray fiber diffraction. J Mol Biol. 1989 Jul 20;208(2):307–325. doi: 10.1016/0022-2836(89)90391-4. [DOI] [PubMed] [Google Scholar]
- Nejidat A., Beachy R. N. Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses. Mol Plant Microbe Interact. 1990 Jul-Aug;3(4):247–251. doi: 10.1094/mpmi-3-247. [DOI] [PubMed] [Google Scholar]
- Osbourn J. K., Watts J. W., Beachy R. N., Wilson T. M. Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein. Virology. 1989 Sep;172(1):370–373. doi: 10.1016/0042-6822(89)90143-8. [DOI] [PubMed] [Google Scholar]
- Powell P. A., Sanders P. R., Tumer N., Fraley R. T., Beachy R. N. Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences. Virology. 1990 Mar;175(1):124–130. doi: 10.1016/0042-6822(90)90192-t. [DOI] [PubMed] [Google Scholar]
- Register J. C., 3rd, Beachy R. N. Resistance to TMV in transgenic plants results from interference with an early event in infection. Virology. 1988 Oct;166(2):524–532. doi: 10.1016/0042-6822(88)90523-5. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siegel A., Hills G. J., Markham R. In vitro and in vivo aggregation of the defective PM2 tobacco mosaic virus protein. J Mol Biol. 1966 Aug;19(1):140–144. doi: 10.1016/s0022-2836(66)80056-6. [DOI] [PubMed] [Google Scholar]
- Tumer N. E., Kaniewski W., Haley L., Gehrke L., Lodge J. K., Sanders P. The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2331–2335. doi: 10.1073/pnas.88.6.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu X. J., Beachy R. N., Wilson T. M., Shaw J. G. Inhibition of uncoating of tobacco mosaic virus particles in protoplasts from transgenic tobacco plants that express the viral coat protein gene. Virology. 1990 Dec;179(2):893–895. doi: 10.1016/0042-6822(90)90163-l. [DOI] [PubMed] [Google Scholar]
- Yusibov V., Loesch-Fries L. S. High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8980–8984. doi: 10.1073/pnas.92.19.8980. [DOI] [PMC free article] [PubMed] [Google Scholar]
