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
1997-10-06
2002-01-08
McElwain, Elizabeth F. (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, C435S419000, C435S468000, C435S469000, C800S288000, C800S301000, C800S294000, C800S317000
Reexamination Certificate
active
06337431
ABSTRACT:
FIELD OF THE INVENTION
This invention is related to the genetic engineering of plants and to a means and method for conferring a plurality of traits, including resistance to viruses, to a plant using a vector encoding a plurality of genes, such as coat protein genes, protease genes, or replicase genes.
BACKGROUND OF THE INVENTION
Many agriculturally important crops are susceptible to infection by plant viruses, which can seriously damage a crop, reduce its economic value to the grower, and increase its cost to the consumer. Attempts to control or prevent infection of a crop by a plant virus have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the genetic material which provides the protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. “Resistant” is the opposite of “susceptible,” and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
Potyviruses are a distinct group of plant viruses which are pathogenic to various crops, and which demonstrate cross-infectivity between plant members of different families. Potyviruses include watermelon mosaic virus-2 (WMV-2); papaya ringspot virus strains papaya ringspot and watermelon mosaic I (PRV-p and PRV-w), two closely related members of the plant potyvirus group which were at one time classified as distinct virus types, but are presently classified as different strains of the same virus; zucchini yellow mosaic virus (ZYMV); potato virus Y; tobacco etch and many others. For example, see Table I of published European patent application 578,627.
These viruses consist of flexous, filamentous particles of dimensions approximately 780×12 nanometers. The viral particles contain a single-stranded RNA genome containing about 10,000 nucleotides of positive (+, coding, or sense) polarity. Translation of the RNA genome of potyviruses shows that the RNA encodes a single large polyprotein of about 330 kD. This polyprotein contains several proteins, one of which is a 49 kD protease that is specific for the cleavage of the polyprotein into at least six (6) other peptides. These proteins can be found in the infected plant cell and form the necessary components for viral replication. One of the proteins contained within this polyprotein is a 35 kD capsid or coat protein which coats and protects the viral RNA from degradation. Another protein is the nuclear inclusion protein, also referred to as replicase, which is believed to function in the replication of the viral RNA. In the course of a potyviral infection, the replicase protein (60 kDa, also referred to as the nuclear inclusion B protein) and the protease protein (50 kDa, also referred to as the nuclear inclusion I or nuclear inclusion A protein) are posttranslationally transported across the nuclear membrane into the nucleus of the plant cell at the later stages of viral infection and accumulate to high levels.
Generally, the coat protein gene is located at the 3′-end of the RNA, just prior to a stretch of terminal adenine nucleotide residues (200 to 300 bases). The location of the 49 Kd protease gene appears to be conserved in these viruses. In the tobacco etch virus, the protease cleavage site has been determined to be the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. Conservation of these dipeptides as the cleavage sites in these viral polyproteins is apparent from the sequences of the above-listed potyviruses.
Expression of the coat protein genes from tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus. Some evidence of heterologous protection has also been reported. For example, Namba et al.,
Phytopathology,
82, 940 (1992) report that expression of coat protein genes from watermelon mosaic virus-2 or zucchini yellow mosaic virus in transgenic tobacco plants conferred protection against six other potyviruses: bean yellow mosaic virus, potato virus Y, pea mosaic virus, clover yellow vein virus, pepper mottle virus and tobacco etch virus. Stark et al.,
Biotechnology,
1, 1257 (1989) report that expression of the potyvirus soybean mosaic virus in transgenic plants provided protection against two serologically unrelated potyviruses: tobacco etch virus and potato virus Y.
However, expression of a preselected coat protein gene does not reliably confer heterologous protection to a plant. For example, transgenic squash plants containing the CMV-C coat protein gene and which have been shown to be resistant to CMV-C strain, are not protected against several highly virulent strains of CMV, including CMV-V-27 and CARNA-5. Thus, a need exists for improved methods to impart potyvirus resistance to plants.
SUMMARY OF THE INVENTION
The present invention provides a recombinant chimeric DNA molecule comprising a plurality of DNA sequences each of which comprises a promoter operably linked to a DNA sequence which encodes a virus-associated protein, such as a coat protein (cp), a protease, or a replicase, wherein said DNA sequences are expressed in virus-susceptible plant cells transformed with said recombinant DNA molecule to impart resistance to infection by each of said viruses. Preferably, the DNA sequences are linked in tandem, i.e., exist in head to tail orientation relative to one another. Also, preferably substantially equal levels of resistance to infection by each of said viruses occurs in plant cells transformed with said plurality of DNA sequences.
Preferably, each DNA sequence is also linked to a 3′ non-translated DNA sequence which functions in plant cells to cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3′ end of the transcribed mRNA sequences. Preferably, the virus is a plant-associated virus, such as a potyvirus.
Thus, the present DNA molecule can be employed as a chimeric recombinant “expression construct,” or “expression cassette” to prepare transgenic plants that exhibit increased resistance to infection by at least two plant viruses, such as potyviruses. The present cassettes also preferably comprise at least one selectable marker gene or reporter gene which is stably integrated into the genome of the transformed plant cells in association with the viral genes. The selectable marker and/or reporter genes facilitate identification of transformed plant cells and plants. Preferably, the virus gene array is flanked by two or more selectable marker genes, reporter genes or a combination thereof. Another aspect of the present invention is a method of preparing a virus-resistant plant, such as a dicot, comprising:
(a) transforming plant cells with a chimeric recombinant DNA molecule comprising a plurality of DNA sequences, each comprising a promoter functional in said plant cells, operably linked to a DNA sequence, which encodes a protein associated with a virus which is capable of infecting said plant;
(b) regenerating said plant cells to provide a differentiated plant;
and
(c) identifying a transformed plant which expresses the DNA sequences so as to render the plant resistant to infection by said viruses, preferably at substantially equal levels of resistance to infection by each virus.
Yet another object of the present invention is to provide a method for providing resistance to infection by viruses in a susceptible Cucurbitaceae plant whic
Carney Kim J.
Deng Rosaline Z.
McMaster Russell J.
Quemada Hector D.
Reynolds John F.
Gardner Carton & Douglas
McElwain Elizabeth F.
Mehta Ashwin
Mueller Lisa V.
Seminis Vegetable Seeds Inc.
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