Plant Bioscience Limited

Plant Biosciences Limited

David Charles Baulcombe

Andrew John Hamilton

Olivier Voinnet

Susan Mary Angell

Carsten Werner Lederer

Ana Montserrat Martin-Hernandez

Frank Giles Ratcliff

Disclosed are a variety of methods for achieving enhanced expression from a target nucleotide sequence in a plant e.g. comprising the step of transiently introducing into a tissue of a plant (e.g. a leaf) a first nucleic acid comprising the target nucleotide sequence and a second nucleic acid encoding a Post Transcriptional Gene Silencing (PTGS) suppressor protein (preferably of viral or plant origin), wherein the first and second nucleic acids are comprised within a single binary vector, construct, or the first and second nucleic acid sequences are comprised within a first binary vector and a second binary vector construct respectively. The plant tissue may then be harvested for the protein. Such methods can give much higher levels of gene expression than are obtainable using stable transgenes, or certain replicating vectors. Also disclosed are specific PTGS suppressor proteins: potato virus X (pvx), p25 protein; african cassava mosaic virus (acmv) AC2 protein; rice yellow mottle virus (rymv) P1 protein; tomato bushy stunt virus (tbsv) 19K protein; plus variants of these. These suppressors may be used in any PTRS context, including the enhancement of transient expression systems.

Enhanced transgene expression by co-expression with a suppressor of post-transcriptional gene silencing (PTGS)

Methods are disclosed for screening for the occurrence of gene silencing (e.g., post transcriptional gene silencing) in an organism. Also provided are methods for isolating silencing agents so identified.

Gene silencing

Cells that have been exogenously supplied with short RNA molecules to effect posttranscriptional gene silencing are described.

Short RNA producing gene silencing in cells

The invention is directed to compositions for gene silencing by providing short RNA molecules to cells.

RNA molecules and vectors for gene silencing

Methods are disclosed for gene silencing (e.g. post transcriptional gene silencing) in an organism using small RNA molecules.

The invention relates to short RNA molecules of about 25 nucleotides in length, which comprise antisense RNA complementary to a targeted mRNA and corresponding sense RNA molecules. These short RNA molecules are effective in post-transcriptional gene silencing (PTGS).

A method to silence a gene in cells by post-transcriptional gene silencing is described which method employs short RNA molecules (SRMs). SRMs are short sense RNA molecules (SSRMs) and short antisense RNA molecules (SARMs) which SARMs are complementary to a region of a target RNA transcribed from a gene to be silenced and said SSRMs correspond to the sequence of the target RNA.

Methods are disclosed for screening of the occurrence of gene silencing (e.g. post transcriptional gene silencing) in an organism. Also provided are methods for isolating silencing agents so identified.

Methods of detecting silencing mammalian cells

Disclosed are a variety of methods for achieving enhanced expression from a target nucleotide sequence in a plant e.g. comprising the step of transiently introducing into a tissue of a plant (e.g. a leaf) a first nucleic acid comprising the target nucleotide sequence and a second nucleic acid encoding a Post Transcriptional Gene Silencing (PTGS) suppressor protein (preferably of viral or plant origin), wherein the first and second nucleic acids are comprised within a single binary vector construct, or the first and second nucleic acid sequences are comprised within a first binary vector and a second binary vector construct respectively. The plant tissue may then be harvested for the protein. Such methods can give much higher levels of gene expression than are obtainable using stable transgenes, or certain replicating vectors. Also disclosed are specific PTGS suppressor proteins: potato virus X (pvx) p25 protein; african cassava mosaic virus (acmv) AC2 protein; rice yellow mottle virus (rymv) P1 protein; tomato bushy stunt virus (tbsv) 19K protein; plus variants of these. These suppressors may be used in any PTRS context, including the enhancement of transient expression systems.

The invention presents DNA constructs comprising a promoter operably linked to DNA which can be transcribed in a plant cell to an RNA transcript, wherein the RNA transcript comprises plant virus sequence from an RNA virus which confers on the RNA transcript the ability to replicate in the cytoplasm of the plant cell, wherein the transcript lacks all or part of the viral genome not required for replication in the cytoplasm, and further comprises at least one targeting sequence which is foreign to the plant virus sequence and causes post-transcriptional gene silencing of one or more target genes. The invention also presents methods to use the DNA construct to cause post-transcriptional gene silencing of a target gene in a plant.

Methods and DNA constructs for gene silencing in transgenic plants

Disclosed are methods for silencing a target nucleotide sequence (preferably representing one or more endogenous genes, preferably in a systemic fashion) which is present in a first part of the plant, which method comprises transiently introducing into the cytoplasm of a cell in a second part of the plant, which cell comprises a nucleic acid encoding the target sequence and which is remote from said first part of the plant, a nucleic acid construct.

Gene silencing methods

Disclosed are nucleic acid vectors which comprise: (a) a transfer nucleotide sequence comprising (i) a plant active promoter, operably linked to (ii) a recombinant tobacco rattle virus (TRV) cDNA (preferably derived from TRV RNA2) which includes at least cis acting elements permitting replication of the cDNA; a subgenomic promoter operably linked to a sequence encoding a TRV coat protein; and a heterologous nucleotide sequence which is foreign to the virus;(b) border sequences which permit the transfer of the transfer nucleotide sequence into a plant genome. Such vectors may be used as expression vectors or for achieving viral induced gene silencing (VIGS) of a target gene, wherein the heterologous nucleotide sequence is a targeting sequence which corresponding to that gene. Example vectors include pTV00 and vectors which are derived from PTV00 and have the characteristics thereof. Also disclosed are associated processes, methods, viruses or viral particle, kits, host cells and plant tissues.

Growing community of inventors

Norfolk, United Kingdom

David Charles Baulcombe