Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing Free

Abstract

Helper component proteinase (HC-Pro) of is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using infiltration in transgenic plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.82200-0
2007-03-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/88/3/1005.html?itemId=/content/journal/jgv/10.1099/vir.0.82200-0&mimeType=html&fmt=ahah

References

  1. Anandalakshmi R., Pruss G. J., Ge X., Marathe R., Mallory A. C., Smith T. H., Vance V. B. 1998; A viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95:13079–13084 [CrossRef]
    [Google Scholar]
  2. Brigneti G., Voinnet O., Li W. X., Ji L. H., Ding S. W., Baulcombe D. C. 1998; Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana . EMBO J 17:6739–6746 [CrossRef]
    [Google Scholar]
  3. Cao Y., Hallet B., Sherratt D. J., Hayes F. 1997; Structure-function correlations in the XerD site-specific recombinase revealed by pentapeptide scanning mutagenesis. J Mol Biol 274:39–53 [CrossRef]
    [Google Scholar]
  4. Carrington J. C., Cary S. M., Parks T. D., Dougherty W. G. 1989; A second proteinase encoded by a plant potyvirus genome. EMBO J 8:365–370
    [Google Scholar]
  5. Cronin S., Verchot J., Haldeman-Cahill R., Schaad M. C., Carrington J. C. 1995; Long-distance movement factor: a transport function of the potyvirus helper component-proteinase. Plant Cell 7:549–559 [CrossRef]
    [Google Scholar]
  6. Cunningham B. C., Wells J. A. 1989; High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244:1081–1085 [CrossRef]
    [Google Scholar]
  7. Dolja V. V., Herndon K. L., Pirone T. P., Carrington J. C. 1993; Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene. J Virol 67:5968–5975
    [Google Scholar]
  8. Dunoyer P., Lecellier C. H., Parizotto E. A., Himber C., Voinnet O. 2004; Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16:1235–1250 [CrossRef]
    [Google Scholar]
  9. Fransen M., Vastiau I., Brees C., Brys V., Mannaerts G. P., Van Veldhoven P. P. 2005; Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis. J Mol Biol 346:1275–1286 [CrossRef]
    [Google Scholar]
  10. González-Jara P., Atencio F. A., Martínez-García B., Barajas D., Tenllado F., Díaz-Ruíz J. R. 2005; A single amino acid mutation in the plum pox virus helper component-proteinase gene abolishes both synergistic and RNA silencing suppression activities. Phytopathology 95:894–901 [CrossRef]
    [Google Scholar]
  11. Haapa S., Taira S., Heikkinen E., Savilahti H. 1999; An efficient and accurate integration of mini-Mu transposons in vitro : a general methodology for functional genetic analysis and molecular biology applications. Nucleic Acids Res 27:2777–2784 [CrossRef]
    [Google Scholar]
  12. Hallet B., Sherratt D. J., Hayes F. 1997; Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein. Nucleic Acids Res 25:1866–1867 [CrossRef]
    [Google Scholar]
  13. Hamilton A. J., Voinnet O., Chappell L., Baulcombe D. C. 2002; Two classes of short interfering RNA in RNA silencing. EMBO J 21:4671–4679 [CrossRef]
    [Google Scholar]
  14. Haseloff J., Siemering K. R., Prasher D. C., Hodge S. 1997; Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci U S A 94:2122–2127 [CrossRef]
    [Google Scholar]
  15. Hayes F. 2003; Transposon-based strategies for microbial functional genomics and proteomics. Annu Rev Genet 37:3–29 [CrossRef]
    [Google Scholar]
  16. Johansen L. K., Carrington J. C. 2001; Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium -mediated transient expression system. Plant Physiol 126:930–938 [CrossRef]
    [Google Scholar]
  17. Kasschau K. D., Carrington J. C. 1998; A counterdefensive strategy of plant viruses: suppression of post-transcriptional gene silencing. Cell 95:461–470 [CrossRef]
    [Google Scholar]
  18. Kasschau K. D., Carrington J. C. 2001; Long-distance movement and replication maintenance functions correlate with silencing-suppression activity of potyviral HC-Pro. Virology 285:71–81 [CrossRef]
    [Google Scholar]
  19. Kasschau K. D., Cronin S., Carrington J. C. 1997; Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase. Virology 228:251–262 [CrossRef]
    [Google Scholar]
  20. Kasschau K. D., Xie Z., Allen E., Llave C., Chapman E. J., Krizan K. A., Carrington J. C. 2003; P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4:205–217 [CrossRef]
    [Google Scholar]
  21. Klein P. G., Klein R. R., Rodriguez-Cerezo E., Hunt A. G., Shaw J. G. 1994; Mutational analysis of the tobacco vein mottling virus genome. Virology 204:759–769 [CrossRef]
    [Google Scholar]
  22. Lakatos L., Csorba T., Pantaleo V., Chapman E. J., Carrington J. C., Liu Y. P., Dolja V. V., Calvino L. F., Lopez-Moya J. J., Burgyan J. 2006; Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25:2768–2780 [CrossRef]
    [Google Scholar]
  23. Llave C., Kasschau K. D., Carrington J. C. 2000; Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci U S A 97:13401–13406 [CrossRef]
    [Google Scholar]
  24. Loon C. L. 1975; Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var ‘Samsun’ and ‘Samsun’ (NN. IV Similarity of qualitative changes of specific proteins after infection with different viruses and their relationship to acquired resistance). Virology 67:566–575 [CrossRef]
    [Google Scholar]
  25. Maiss E., Timpe U., Brisske A., Jelkmann W., Casper R., Himmler G., Mattanovich D., Katinger H. W. D. 1989; The complete nucleotide sequence of plum pox virus RNA. J Gen Virol 70:513–524 [CrossRef]
    [Google Scholar]
  26. Maiss E., Timpe U., Brisske-Rode A., Lesemann D. E., Casper R. 1992; Infectious in vivo transcripts of plum pox potyvirus full-length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter. J Gen Virol 73:709–713 [CrossRef]
    [Google Scholar]
  27. Mallory A. C., Ely L., Smith T. H., Marathe R., Anandalakshmi R., Fagard M., Vaucheret H., Pruss G., Bowman L., Vance V. B. 2001; HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13:571–583 [CrossRef]
    [Google Scholar]
  28. Mallory A. C., Reinhart B. J., Bartel D., Vance V. B., Bowman L. H. 2002; A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco. Proc Natl Acad Sci U S A 99:15228–15233 [CrossRef]
    [Google Scholar]
  29. Mérai Z., Kerenyi Z., Molnar A., Barta E., Valoczi A., Bisztray G., Havelda Z., Burgyan J., Silhavy D. 2005; Aureusvirus P14 is an efficient RNA silencing suppressor that binds double-stranded RNAs without size specificity. J Virol 79:7217–7226 [CrossRef]
    [Google Scholar]
  30. Merits A., Guo D., Saarma M. 1998; VPg, coat protein and five non-structural proteins of potato A potyvirus bind RNA in a sequence-unspecific manner. J Gen Virol 79:3123–3127
    [Google Scholar]
  31. Oh C. S., Carrington J. C. 1989; Identification of essential residues in potyvirus proteinase HC-Pro by site-directed mutagenesis. Virology 173:692–699 [CrossRef]
    [Google Scholar]
  32. Poussu E., Vihinen M., Paulin L., Savilahti H. 2004; Probing the alpha-complementing domain of E. coli beta-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition. Proteins 54:681–692 [CrossRef]
    [Google Scholar]
  33. Pruss G., Ge X., Shi X. M., Carrington J. C., Bowman Vance V. 1997; Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9:859–868 [CrossRef]
    [Google Scholar]
  34. Qu F., Ren T., Morris T. J. 2003; The coat protein of Turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step. J Virol 77:511–522 [CrossRef]
    [Google Scholar]
  35. Raccah B., Huet H., Blanc S. 2001; Potyviruses. In Virus-Insect-Plant Interactions pp 181–206 Edited by Harris K., Duffus J. E., Smith O. P. San Diego, CA: Academic Press;
    [Google Scholar]
  36. Revers F., Le Gall O., Candresse T., Maule A. J. 1999; New advances in understanding the molecular biology of plant/potyvirus interactions. Mol Plant Microbe Interact 12:367–376 [CrossRef]
    [Google Scholar]
  37. Riedel D., Lesemann D. E., Maiss E. 1998; Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus. Arch Virol 143:2133–2158 [CrossRef]
    [Google Scholar]
  38. Sáenz P., Quiot L., Quiot J. B., Candresse T., Garcia J. A. 2001; Pathogenicity determinants in the complex virus population of a Plum pox virus isolate. Mol Plant Microbe Interact 14:278–287 [CrossRef]
    [Google Scholar]
  39. Silhavy D., Burgyan J. 2004; Effects and side-effects of viral RNA silencing suppressors on short RNAs. Trends Plant Sci 9:76–83
    [Google Scholar]
  40. Silhavy D., Molnar A., Lucioli A., Szittya G., Hornyik C., Tavazza M., Burgyan J. 2002; A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21:3070–3080 [CrossRef]
    [Google Scholar]
  41. Shi X. M., Miller H., Verchot J., Carrington J. C., Vance V. B. 1997; Mutations in the region encoding the central domain of helper component proteinase (HC-Pro) eliminate potato virus X/potyviral synergism. Virology 231:35–42 [CrossRef]
    [Google Scholar]
  42. Thompson J. D., Gibson T. B., Plewniak F., Jeanmougin F., Higgins D. G. 1997; The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 [CrossRef]
    [Google Scholar]
  43. Urcuqui-Inchima S., Maia I. G., Arruda P., Drugeon G., Haenni A.-L., Bernardi F. 2000; Deletion mapping of the potyviral helper component-proteinase reveals two regions in RNA binding. Virology 268:104–111 [CrossRef]
    [Google Scholar]
  44. Urcuqui-Inchima S., Haenni A. L., Bernardi F. 2001; Potyvirus proteins: a wealth of functions. Virus Res 74:157–175 [CrossRef]
    [Google Scholar]
  45. Vance V. B. 1991; Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology 182:486–494 [CrossRef]
    [Google Scholar]
  46. Vance V. B., Berger P. H., Carrington J. C., Hunt A. G., Shi X. M. 1995; 5′ proximal potyviral sequences mediate potato virus X/potyviral synergistic disease in transgenic tobacco. Virology 206:583–590 [CrossRef]
    [Google Scholar]
  47. Voinnet O., Pinto V. M., Baulcombe D. C. 1999; Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96:14147–14152 [CrossRef]
    [Google Scholar]
  48. Voinnet O., Lederer C., Baulcombe D. C. 2000; A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana . Cell 103:157–167 [CrossRef]
    [Google Scholar]
  49. Yang S., Ravelonandro M. 2002; Molecular studies of the synergistic interactions between Plum pox virus HC-Pro protein and potato virus X. Arch Virol 147:2301–2312 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.82200-0
Loading
/content/journal/jgv/10.1099/vir.0.82200-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed