Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
2001-10-31
2004-08-17
Mehta, Ashwin (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C435S320100, C435S468000, C800S260000
Reexamination Certificate
active
06777588
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for producing transgenic cereal plants resistant to Barley Yellow Dwarf Virus, particularly in the presence of co-infecting Cereal Yellow Dwarf Virus, by stably integrating into the cells of the transgenic plant a chimeric gene comprising a DNA region operably linked to plant expressible promoter in such a way that a RNA molecule may be transcribed from the DNA region, the RNA molecule comprising both sense and antisense RNA capable of pairing and forming a double stranded RNA molecule or hairpin RNA.
DESCRIPTION OF RELATED ART
Barley yellow dwarf virus-PAV (BYDV-PAV) is the most serious and widespread virus of cereals worldwide. The barley yellow dwarf virus (BYDV), also called red leaf in oats, can infect barley, oats, rye and wheat as well as numerous species of grasses. It occurs in most parts of the world and is considered the most common viral disease of cereal crops.
BYDV (and CYDV) have been reported to cause cereal disease in over 50 countries. In almost all cases where the species has been determined, the major losses have been due to BYDV-PAV (Barker & Waterhouse, 1999). Lost production due to BYDV averages about 15% in barley, 17% in wheat and 25% in oats (Lister and Ranieri, 1995). BYDV infection can affect plant height, grain size and grain quality. For example, BYDV infection of barley can reduce the grain quality such that it is suitable only for animal feed rather than malting.
BYDV is transmitted by several species of aphids. As the aphids (winged or wingless) feed on the cereal crop, they transmit the virus through their mouthparts. The aphids can remain infectious for life, which is around 40 days.
Disease symptoms vary with the host species and the stage of crop development. Infections at the seedling stage may result in death or dwarfing as well as sterile heads. Leaves turn yellow from the tip down, along the leaf margins or in blotchy patches. Infected barley leaves, particularly flag leaves, turn bright yellow. In oats, the leaves may turn from red to purple. Discolored areas enlarge and progress to the base of the plant. Heads may be wholly or partially sterile. There may also be an increase or decrease of tillers produced by infected plants. Cereal plants infected early in the season may be shaded out by healthy or late infected surrounding plants. Winter wheat seedlings may be 100 percent infected with BYDV before freeze-up in the fall. BYDV affects yields by stunting, reduced tillering, sterility, and failure to fill kernels.
Natural resistance genes against this luteovirus give inadequate control. Sources of natural resistance to BYDV and CYDV are rare (for reviews see Barker and Waterhouse 1999; Burnett et al., 1995). In barley, the Yd2 gene (Paltridge et al., 1998), originally identified in Ethiopian concessions (Rasmussin and Schaller, 1959; Schaller et al., 1964), can confer resistance against BYDV-PAV, but its effectiveness varies depending on the genetic background of the plant and growth conditions (Schaller, 1984; Larkin et al., 1991). The Bdv1 gene confers some tolerance to BYDV (Singh et al, 1993) and has been introduced into some wheat cultivars, such as Anza. However, BYDV replicates and causes symptoms and yield loss in plants containing either the Yd2 or Bdv1 genes.
Previous attempts to introduce synthetic resistance into cereals have produced variable results. Virus resistance in plants containing virus-derived transgenes, usually by the expression of functional or dysfunctional coat protein, movement or polymerase genes, has been widely reported (for review see Waterhouse & Upadhyaya 1998) and attempts have been made to produce transgenic plants with resistance to a few different luteoviruses, by expression of coat protein and polymerase genes.
McGrath et al. (1997) transformed oat and barley plants with transgenes derived from the coat protein genes of BYDV-PAV and CYDV-RPV and obtained some resistant plants. However, this resistance was not stable.
In another study, Koev et al. (1998) transformed oat plants with the 5′half of the BYDV-PAV genome and found one line that after inoculation with BYDV-PAV showed disease symptoms but recovered and produced seed. Although BYDV resistance in the progeny of this line was inherited, the levels of resistance varied greatly among individual plants, ranging from substantial to undetectable.
Waterhouse et al., 1998, Wang & Waterhouse, 2000 and Smith et al., 2000 describe that virus immunity and posttranscriptional gene silencing (PTGS) can be induced in plants using transgenes that encode double stranded (ds) or self-complementary “hairpin” (hp) RNA.
WO 98/53083 describes constructs and methods for enhancing the inhibition of a target gene within an organism involve inserting into the gene-silencing vector an inverted repeat sequence for all or part of a polynucleotide region within the vector. The inverted repeat sequence may be a synthetic polynucleotide sequence or comprise a modified natural phenotype.
WO 99/32619 provides a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with a double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequence of the duplex region of the RNA and of a portion of the target gene are identical.
WO 99/49029 relates to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. Recombinant DNA technology is used to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Synthetic genes and genetic constructs which are capable of repressing, delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.
WO 99/53050 provides methods and means for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprise simultaneously both sense and antisense nucleotide sequence.
WO 99/61631 relates to methods to alter the expression of a target gene in a plant using sense and antisense RNA fragments of the gene. The sense and antisense RNA fragments are capable of pairing and forming a double-stranded RNA molecule, thereby altering the expression of the gene. This publication also relates to plants, their progeny and seeds derived thereof, obtained using the methods described.
WO 00/49035 discloses a method for silencing the expression of an endogenous gene in a cell, the method involving overexpressing in the cell a nucleic acid molecule of the endogenous gene, wherein the overexpression of the nucleic acid molecule of the endogenous gene and the antisense molecule in the cell silences the expression of the endogenous gene.
SUMMARY AND OBJECTS OF THE INVENTION
The invention provides DNA molecules comprising a plant-expressible promoter operably linked to a DNA region which when transcribed in the cells of a cereal plant yields an RNA molecule comprising a first nucleotide sequence of at least 19 bp having at least 70% nucleotide sequence identity to the sense nucleotide sequence of a BYDV isolate encoding the polymerase gene and a second nucleotide sequence of at least 19 bp having at least 70% nucleotide sequence identity to the complement of the sense nucleotide sequence of an RNA dependent RNA polymerase (hereinafter referred to as “polymerase”) gene of a BYDV isolate, such as but not limited to a polymerase comprising the nucleotide sequence of SEQ ID No 1 from the nucleotide at position 1 to the nucleotide at
Abbott David
Wang Ming-Bo
Waterhouse Peter
Burns Doane Swecker & Mathis L.L.P.
Mehta Ashwin
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