Papaya ringspot virus NIa protease gene

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance

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800288, 800294, 536 232, 536 2372, 536 241, 4352523, 4353701, 435468, 435469, A01H 100

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060463848

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BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to a protease gene derived from papaya ringspot virus. More specifically, the invention relates to the genetic engineering of plants and to a method for conferring viral resistance to a plant using an expression cassette encoding papaya ringspot virus PRV FLA83 W or PRV USA P-type (HA attenuated) protease.


BACKGROUND OF THE INVENTION

Many agriculturally important crops are susceptible to infection by plant viruses, particularly papaya ringspot virus, 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 such as papaya ringspot 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. Generally, a potyvirus is a single-stranded RNA virus that is surrounded by a repeating protein monomer, which is termed the coat protein (CP). The majority of the potyviruses are transmitted in a nonpersistent manner by aphids. As can be seen from the wide range of crops affected by potyviruses, the host range includes such diverse families of plants as Solanaceae, Chenopodiaceae, Gramineae, Compositae, Leguminosae, Dioscroeaceae, Cucurbitaceae, and Caricaceae. Potyviruses include watermelon mosaic virus II (WMVII), zucchini yellow mosaic virus (ZYMV), potato virus Y, tobacco etch and many others.
Another potyvirus of economic significance is papaya ringspot virus (PRV). Two groups of PRV have been identified: the "P" or "papaya ringspot" type infects papayas; and the "W" or "watermelon" type infects cucurbits, e.g., squash, but it is unable to infect papaya. Thus, these two groups can be distinguished by host range differences.
The potyviruses consist of flexous, filamentous particles of dimensions approximately 780.times.12 nanometers. The viral particles contain a single-stranded positive polarity RNA genome containing about 10,000 nucleotides. 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; these include the coat protein, nuclear inclusion proteins NIa and NIb, cytoplasmic inclusion protein (CI), and other proteases and movement proteins (see FIG. 1). These proteins are found in the infected plant cell and form the necessary components for viral replication. One of the proteins contained in the polyprotein is a 35 kD capsid or coat protein which coats and protects the viral RNA from degradation. One of the nuclear inclusion proteins, NIb, is an RNA replicase component and is thought to have polymerase activity. CI, a second inclusion protein, is believed to participate in the replicase complex and have a helicase activity. NIa, a third inclusion protein, has a protease activity. In the course of potyvirus infection, NIa and NIb are translationally transported across the nuclear membrane into the nucleus of the infected plant cell at the l

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