Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs.

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with several transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Resistance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haploid plant and isogenic for the PVY transgenes expressing untranslatable sense RNA, displayed different levels of PVY resistance. Lines with actively transcribed, methylated transgene sequences had low steady state levels of transgene transcript and a virus-resistant phenotype. These results are discussed within the context of sense suppression in plants.

[1]  J. Mol,et al.  More about co-suppression , 1991 .

[2]  J. Riechmann,et al.  Highlights and prospects of potyvirus molecular biology. , 1992, The Journal of general virology.

[3]  A. Hoekema,et al.  A small-scale procedure for the rapid isolation of plant RNAs. , 1989, Nucleic acids research.

[4]  W. Dougherty,et al.  Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance. , 1993, The Plant cell.

[5]  R. Goldbach,et al.  Characterization of RNA–Mediated Resistance to Tomato Spotted Wilt Virus in Transgenic Tobacco Plants , 1992, Bio/Technology.

[6]  W. Dougherty,et al.  Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. , 1992, Virology.

[7]  H. L. Sänger,et al.  RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties. , 1993, The Journal of biological chemistry.

[8]  M. Matzke,et al.  Genomic imprinting in plants: parental effects and trans-inactivation phenomena , 1993 .

[9]  F. Eckstein,et al.  The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. , 1985, Nucleic acids research.

[10]  H. L. Sänger,et al.  RNA-directed de novo methylation of genomic sequences in plants , 1994, Cell.

[11]  F. Eckstein,et al.  The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. , 1985, Nucleic acids research.

[12]  J. Mol,et al.  Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. , 1990, The Plant cell.

[13]  S. Rogers,et al.  Extraction of DNA from plant tissues , 1989 .

[14]  J. Haldane,et al.  Basic Genetics , 1964, Nature.

[15]  E. Koonin,et al.  Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. , 1993, Critical reviews in biochemistry and molecular biology.

[16]  C. Napoli,et al.  Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. , 1990, The Plant cell.

[17]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[18]  V. Dolja,et al.  Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. , 1994, The EMBO journal.

[19]  D. Inzé,et al.  Suppression of beta‐1,3‐glucanase transgene expression in homozygous plants. , 1992, The EMBO journal.

[20]  M. Kasperbauer,et al.  Reconstitution of Diploids from Leaf Tissue of Anther-derived Haploids in Tobacco 1 , 1972 .

[21]  C. H. T. Cate,et al.  Differential methylation and expression of the β-glucuronidase and neomycin phosphotransferase genes in transgenic plants of potato cv. Bintje , 1993 .

[22]  T. Wilson.  Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D. Gonsalves,et al.  Different Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses , 1993, Bio/Technology.

[24]  W. Dougherty,et al.  Translation of potyvirus RNA in a rabbit reticulocyte lysate: identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome. , 1980, Virology.

[25]  E. A. Wernsman,et al.  Maternal Haploids of Nicotiana tabacum L. from Seed , 1979, Science.

[26]  K. Cox.  Analysis of plant gene expression , 1988 .

[27]  D. Grierson,et al.  Does co-suppression of sense genes in transgenic plants involve antisense RNA? , 1991 .

[28]  W. Dougherty,et al.  Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. , 1992, Molecular plant-microbe interactions : MPMI.

[29]  S. A. Johnston,et al.  Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. , 1994, Analytical biochemistry.

[30]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[31]  H. A. Smith,et al.  RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. , 1994, Molecular plant-microbe interactions : MPMI.

[32]  J. Fry,et al.  A simple and general method for transferring genes into plants. , 1985, Science.

[33]  H. L. Sänger,et al.  RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties. , 1993, The Journal of biological chemistry.

[34]  M. Primig,et al.  Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants , 1989, The EMBO journal.

[35]  W. Dougherty,et al.  Pathogen derived resistance to potyviruses: working, but why? , 1993 .

[36]  R. Flavell.  Inactivation of gene expression in plants as a consequence of specific sequence duplication. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[37]  W F Thompson,et al.  Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. , 1993, The Plant cell.

[38]  T. Ogawa.  [Transgenic resistance to plant viruses]. , 1994, Uirusu.