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1. WO2003018809 - ISOLEMENT DE PROTEINES IMPLIQUEES DANS L'EXTINCTION DE GENES POST-TRANSCRIPTIONNELLE ET METHODES D'UTILISATION

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[ EN ]

WHAT IS CLAIMED IS:

1. A method of isolating nucleic acid encoding a plant polypeptide active in PTGS comprising the steps of :
i) selecting a bait nucleic acid which encodes a bait protein active in PTGS in plants or suppressive of PTGS in plants;
ii) preparing a cDNA prey library from a plant wherein the plant exhibits PTGS;
iii) conducting a yeast two-hybrid assay with the bait and prey nucleic acids, wherein prey cDNA that yields a true positive yeast two- hybrid assay result encodes a polypeptide active in PTGS in the plant .

2. The method of claim 1 wherein the bait nucleic
acid comprises a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 3,
SEQ. ID. NO. 5, SEQ. ID. NO. 7, SEQ. ID. NO. 9, and
SEQ. ID. NO. 11.

3. The method of claim 1 wherein the plant is a
monocot .

4. The method of claim 3 wherein the monocot is
selected from the group consisting of sugarcane,
corn, sorghum and rice.

The method of claim 1 further comprising the step of confirming interaction between the bait protein and the polypeptide active in PTGS using a farwestern blot assay.

The method of claim 1 further comprising the step of confirming interaction between the bait protein and the polypeptide active in PTGS using a pull down assay.

The method of claim 1 further comprising the step of confirming interaction between the bait protein and the polypeptide active in PTGS using an in planta assay.

The method of claim 7 wherein the in planta assay is conducted in embryonic calli of the plant.

An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 85% identity with the nucleic acid sequence of SEQ. ID.NO. 1.

0. A polypeptide comprising an amino acid having at least 78% identity with the amino acid sequence of SEQ. ID.NO. 2.

1. An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 40% identity with the nucleic acid sequence of SEQ. ID. O. 3.

2. A polypeptide comprising the amino acid sequence of SEQ. ID.NO. 4.

3. An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 93% identity with the nucleic acid sequence of SEQ. ID.NO. 5.

4. A polypeptide comprising an amino acid having at least 95% identity with the amino acid sequence of SEQ. ID.NO. 6.

5. An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 98% identity with the nucleic acid sequence of SEQ. ID.NO. 7.

6. A polypeptide comprising an amino acid having at least 76% identity with the amino acid sequence of SEQ. ID.NO. 8.

7. An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 94% identity with the nucleic acid sequence of SEQ. ID.NO. 9.

8. A polypeptide comprising an amino acid having at least 97% identity with the amino acid sequence of SEQ. ID.NO. 10.

9. An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 89% identity with the nucleic acid sequence of SEQ. ID.NO. 11.

0. A polypeptide comprising an amino acid having at least 78% identity with the amino acid sequence of SEQ. ID.NO. 12.

1. A transgenic plant cell in which PTGS is
suppressed comprising a nucleic acid having a sequence with at least 85% identity with the nucleic acid sequence of SEQ. ID.NO. 1.

2. A transgenic plant cell in which PTGS is enhanced comprising a nucleic acid having a sequence with at least 40% identity with the nucleic acid
sequence of SEQ. ID.NO. 3.

3. A transgenic plant cell comprising a nucleic acid having a sequence selected from the group
consisting of SEQ. ID.NO. 1, SEQ. ID.NO. 3,
SEQ. ID.NO. 5, SEQ. ID.NO. 7, SEQ. ID.NO. 9, and
SEQ. ID.NO. 11.

4. A method of increasing viral resistance in a plant comprising the steps of :
i) selecting a protein suppressive of PTGS in the plant ; and
ii) transforming the plant with a nucleic acid encoding the PTGS suppressive protein.

5. A method of increasing expression of a transgene in a plant comprising the steps of:
i) selecting a protein active in PTGS in the plant; and
ii) transforming the plant with a nucleic acid encoding the protein active in PTGS.

6. A method of suppressing expression of a native gene in a plant comprising:
i) preparing a vector including a nucleic acid with a sequence of the coding portion of the gene wherein the nucleic acid, upon
transcription, products an mRNA molecule
double stranded in the region corresponding the to the coding portion of the gene; and ii) transforming the plant with the vector.