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Investigation of the mechanism underlying the inhibitory effect of heterologous ras genes in plant cells

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Abstract

The ras genes from yeast and mammalian cells were fused to plant expression promoters, and introduced into plant cells via Agrobacterium, to study their effect on cell growth and development. All introduced ras genes had a strong inhibitory effect on callus and shoot regeneration from plant tissues. This is consistent with earlier findings that heterologous ras genes were highly lethal to protoplasts following direct DNA uptake. These effects could not be reversed by increasing exogenous or endogenous cytokinin levels. These effects were also independent of the v-Ha-ras mutations in functionally important regions of Ras proteins such as effector-binding and membrane-binding sites. Similarly, co-transformation with the genes encoding the Ras-negative regulators, GTPase-activating protein and neurofibromin did not affect the ras inhibitory effect, indicating that the mechanism of ras inhibition of plant cells is not related to normal ras cellular functions. This conclusion was supported by further studies in which ras gene expression was modified using various promoters and antisense constructs. The introduced ras sequences remained fully inhibitory regardless of which promoters (inducible or tissue-specific) or which orientations (sense or antisense) were tested. This strongly suggests that the ras DNA sequence itself, rather than the Ras protein or ras mRNA, is directly involved in the inhibitory effect. The mechanism underlying this novel phenomenon remains unknown. Introduced ras genes may inhibit plant cell growth by inducing co-suppression of unknown endogenous ras or ras-related genes, thereby leading to the arrest of cell growth.

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References

  1. Adari H, Lowy DR, Willumsen BM, Der CJ, McCormick F: Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science 240: 518–521 (1988).

    PubMed  Google Scholar 

  2. Anai T, Hasegawa K, Watanabe Y, Uchimiya H, Ishizaki R, Matsui M: Isolation and analysis of cDNAs encoding small GTP-binding proteins of Arabidopsis thaliana. Gene 108: 259–264 (1991).

    Article  PubMed  Google Scholar 

  3. Ballester R, Marchuk D, Boguski M, Saulino A, Letcher R, Wigler M, Collins F: The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell 63: 851–859 (1990).

    Article  PubMed  Google Scholar 

  4. Barbacid M: ras genes. Annu Rev Biochem 56: 779–827 (1987).

    Article  PubMed  Google Scholar 

  5. Beitel GJ, Clark SG, Horvitz HR: C. elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348: 503–509 (1990).

    Article  PubMed  Google Scholar 

  6. Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).

    PubMed  Google Scholar 

  7. Bourne HR, Sanders RA, McCormick F: The GTPase superfamily: a conserved switch diverse cell functions. Nature 348: 125–132 (1990).

    Article  PubMed  Google Scholar 

  8. Chang EH, Maryak JM, Wei CM, Shih TY, Shober R, Cheung HL, Ellis RW, Hager GL, Scolnick EM, Lowy DR: Functional organization of the Harvey murine sarcoma virus genome. J Virol 35: 76–92 (1980).

    PubMed  Google Scholar 

  9. Clark SG, McGrath JP, Levinson AD: Expression of normal and activated human H-ras cDNA in S. cerevisiae. Mol Cell Biol 5: 2746–2752 (1985).

    PubMed  Google Scholar 

  10. Dalta RSS, Hammerlindl JK, Pelcher LE, Selvaraj G, Crosby WL: Bifunctional gene fusion between neomycin phosphotransferase and B-glucuronidase: a broad spectrum genetic marker for plants. J Cell Biochem Suppl: 14E (R115): 279 (1990).

    Google Scholar 

  11. DeFeo-Jones D, Gonda MA, Young HA, Chang EH, Lowy DR, Scolnick EM, Ellis RW: Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus. Proc Natl Acad Sci USA 78: 3328–3332 (1981).

    PubMed  Google Scholar 

  12. DeFeo-Jones D, Tatchell K, Robinson LC, Sigal IS, Vass WC, Scolnick E: Mammalian and yeast ras gene products: biological function in their heterologous systems. Science 228: 179–184 (1985).

    PubMed  Google Scholar 

  13. Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium melioti. Proc Natl Acad Sci USA 77: 7347–7351 (1980).

    PubMed  Google Scholar 

  14. Ellis RW, DeFeo D, Shih TY, Gonda MA, Young HA, Tsuchida N, Lowy DR, Scolnick EM: The p21 src genes of Harvey and Kirsten sarcoma virus originate from divergent members of a family of normal vertebrate genes. Nature 292: 506–511 (1981).

    PubMed  Google Scholar 

  15. Estruch JJ, Chriqui D, Grossmann K, Schell J, Soena A: The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J 10: 2889–2895 (1991).

    PubMed  Google Scholar 

  16. Fraley RT, Rogers SG, Horsch RB, Eichholtz DA, Flick JS, Fink CL, Hoffman NL, Sanders PR: The SEV system: a new disarmed Ti plasmid vector system for plant transformation. Bio/technology 3: 629/635 (1985).

    Article  Google Scholar 

  17. Gibbs JB, Sigal IS, Poe M, Scolnick EM: Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci USA 81: 5704–5708 (1984).

    PubMed  Google Scholar 

  18. Goring DR, Thomson L, Rothstein S: Transformation of a partial nopaline synthase gene into tobacco suppresses the expression of a resident wild-type gene. Proc Natl Acad Sci USA 88: 1770–1774 (1991).

    PubMed  Google Scholar 

  19. Hamilton DA, Dashe DM, Stinson J, Mascarenhas JP: Characterization of a pollen-specific genomic clone from maize. Sex Plant Reprod 2: 208–212 (1989).

    Article  Google Scholar 

  20. Han M, Sternberg PW: let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a Ras protein. Cell 63: 921–931 (1990).

    Article  PubMed  Google Scholar 

  21. Hilson P, Dewulf J, Delporte F, Installe P, Jasquemin JM, Jacobs M, Negrutiu I: Yeast RAS2 affects cell viability, mitotic division and transient gene expression in Nicotiana species. Plant Mol Biol 14: 669–685 (1990).

    Article  PubMed  Google Scholar 

  22. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 178–180 (1983).

    Google Scholar 

  23. Holsters M, deWaele D, Depicker A, Messens E, vanMontagu M, Schell J: Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163: 181–187 (1978).

    Article  PubMed  Google Scholar 

  24. Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

  25. Jefferson RA: Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).

    Google Scholar 

  26. Jefferson RA, Kavanagh TA, Bevan MW: GUS-fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    PubMed  Google Scholar 

  27. Jones S, Vignals M-L, Broach JR: The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to Ras. Mol Cell Biol 11: 2641–2646 (1991).

    PubMed  Google Scholar 

  28. Jorgensen R: Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotechnol 8: 340–344 (1990).

    Article  PubMed  Google Scholar 

  29. Kamada I, Yamauchi S, Youssefian S, Sano H: Transgenic tobacco plants expressing rgpl, a gene encoding a ras-related GTP-binding protein from rice, show distinct morphological characteristics. Plant J 2: 799–807 (1992).

    Google Scholar 

  30. Kataoka T, Powers S, Cameron S, Fasano O, Goldfarb M, Broach J, Wigler M: Functional homology of mammalian and yeast RAS genes. Cell 40: 19–26 (1985).

    Article  PubMed  Google Scholar 

  31. Lacal JC, Anderson PS, Aaronson SA: Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distinct regions for GTP-binding and transforming activity. EMBO J 5: 679–687 (1986).

    PubMed  Google Scholar 

  32. Linn F, Heidmann I, Saedler H, Meyer P: Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: Role of numbers of integrated gene copies and state of methylation. Mol Gen Genet 222: 329–336 (1990).

    PubMed  Google Scholar 

  33. Liu ZR: Ph.D. Thesis, Cornell University (1992).

  34. Lowy DR, Zhang K, Declue JE, Willumsen BW: Regulation of p21ras activity. Trends Genet 7: 346–351 (1991).

    PubMed  Google Scholar 

  35. Matsui M, Sasamoto S, Kunieda T, Nomura N, Ishizaki R: Cloning of ara, a putative Arabidopsis thaliana gene homologous to the ras-related gene family. Gene 76: 313–319 (1989).

    Article  PubMed  Google Scholar 

  36. Matzke MA, Primig M, Trnovsky J, Matzke AJM: Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8: 643–649 (1989).

    Google Scholar 

  37. Matzke MA, Matzke AJM: Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol 16: 821–830 (1991).

    Article  PubMed  Google Scholar 

  38. McCoy MS, Bargmann CI, Weinberg RA: Human colon carcinoma Ki-ras2 oncogene and its corresponding proto-oncogene. Mol Cell Biol 4: 1577–1582 (1984).

    PubMed  Google Scholar 

  39. McGrath JP, Capon DJ, Goeddel DV, Levinson AD: Comparative biochemical properties of normal and activated human ras p21 protein. Nature 310: 644–649 (1984).

    PubMed  Google Scholar 

  40. Medford JI, Horgan R, EI-Sawi Z, Klee HJ: Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1: 404–413 (1989).

    Article  Google Scholar 

  41. Napoli C, Lemieux C, Jorgensen R: Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289 (1990).

    PubMed  Google Scholar 

  42. Pietrzak M, Shillito RD, Hohn T, Potrykus I: Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucvl Acids Res 14: 5857–5868 (1986).

    Google Scholar 

  43. Powers S, Kataoka T, Fasano O, Goldfarb M, Strathern J, Broach J, Wigler M: Genes in S. cerevisiae encoding protein with domains homologous to the mammalian ras proteins. Cell 36: 607–612 (1984).

    PubMed  Google Scholar 

  44. Reddy EP, Reynolds RK, Santos E, Barbacid M: A point mutation is responsible for the acquisition of transforming properties of the T24 human bladder carcinoma oncogene. Nature 300: 149–152 (1982).

    PubMed  Google Scholar 

  45. Reed SI, Ferguson J, Groppe JC: Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28. Mol Cell Biol 2: 412–425 (1982).

    PubMed  Google Scholar 

  46. Reymond CD, Gomer RH, Mehdy MC, Firtel RA: Developmental regulation of a Dictyostelium gene encoding a protein homologous to mammalian Ras protein. Cell 39: 141–148 (1984).

    Article  PubMed  Google Scholar 

  47. Reymond CD, Gome RH, Nellen W, Theibert A, Devreotes P, Firtel RA: Phenotypic changes induced by a mutated ras gene during development of Dictyostelium transformants. Nature 323: 340–343 (1986).

    PubMed  Google Scholar 

  48. Reymond CD, Nellen W, Firtel RA: Regulated expression of ras gene constructs in Dictyostelium transformants. Proc Natl Acad Sci USA 82: 7005–7009 (1985).

    PubMed  Google Scholar 

  49. Rogers SG, Klee HJ, Horsch RB, Fraley RT: Improved vectors for plant transformation: expression cassette vectors and new selectable Markers. Meth Enzymol 153: 253–277 (1987).

    Google Scholar 

  50. Schena M, Lloyd AM, Davis RW: A steroid-inducible gene expression system for plant cells. Proc Natl Acad Sci USA 88: 10421–10425 (1991).

    PubMed  Google Scholar 

  51. Simon MA, Bowtell DDL, Dodson GS, Laverty TR, Rubin GM: Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67: 701–716 (1991).

    Article  PubMed  Google Scholar 

  52. Smart CM, Scofield SR, Bevan MW, Dyer TA: Delayed leaf senescence in tobacco plants transformed with tmr, a gene for cytokinin production in Agrobacterium. Plant Cell 3: 647–656 (1991).

    Article  PubMed  Google Scholar 

  53. Smigocki AC, Owens LD: Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells. Proc Natl Acad Sci USA 85: 5131–5135 (1988).

    Google Scholar 

  54. Stone JC, Vass WC, Willumsen BM, Lowy DR: p21-ras effector domain mutants constructed by ‘cassette’ mutagenesis. Mol Cell Biol 8: 3565–3569 (1988).

    PubMed  Google Scholar 

  55. Tabin CJ, Bradley SM, Berghman CI, Weinberg RA, Papageorge AG, Scolnick EM, Dhar R, Lowy DR, Chang EH: Mechanism of activation of a human oncogene. Nature 300: 143–149 (1982).

    PubMed  Google Scholar 

  56. Toda T, Uno I, Ishikawa T, Powers S, Kataoka T, Broek D, Cameron S, Broach J, Matsumoto K, Wigler M: In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell 40: 27–36 (1985).

    PubMed  Google Scholar 

  57. van derKrol AR, Mur LA, Beld M, Mol JNM, Stuitje AR: Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291–299 (1990).

    PubMed  Google Scholar 

  58. Willumsen BM, Christensen A, Hubbert NL, Papageorge AG, Lowy DR: The p21 ras C-terminus is required for transformation and membrane association. Nature 310: 583–586 (1984).

    PubMed  Google Scholar 

  59. Willumsen BM, Papageorge AG, Kung HF, Bekesi E, Robins T, Johnsen M, Vass WC, Lowy DR: Mutational analysis of a ras catalytic domain. Mol Cell Biol 6: 2646–2654 (1986).

    PubMed  Google Scholar 

  60. Xu G, Lin B, Tanaka K, Dunn D, Wood D, Gesteland R, White R, Weiss R, Tamanoi F: The catalytic domain of the neurofibromatosis type1 gene product stimulates ras GTPase and complements ira mutation of S. cerevisiae. Cell 63: 835–841 (1990).

    Article  PubMed  Google Scholar 

  61. Zhang K, DeClue JE, Vass WC, Papageorge AG, McCormick F, Lowy DR: Suppression of c-ras transformation by GTPase-activating protein. Nature 346: 754–756 (1990).

    Article  PubMed  Google Scholar 

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  1. Zong R. Liu

    Present address: Department of Biological Sciences, Dartmouth College, 03755, Hanover, NH, USA

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  1. Department of Horticultural Sciences, Cornell University, 14456, Geneva, NY, USA

    Zong R. Liu & John C. Sanford

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Liu, Z.R., Sanford, J.C. Investigation of the mechanism underlying the inhibitory effect of heterologous ras genes in plant cells. Plant Mol Biol 22, 751–765 (1993). https://doi.org/10.1007/BF00027362

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