Functional viral vectors for the overexpression or extinction of particular genes in plants, and applications thereof
View Patent ↗The invention relates to the use of genes which, in plants, encode proteins with a functional diversity in terms of silencing, comprising the selection of the gene with the level of effectiveness in order to construct a plant viral vector having the function of overexpressing or silencing particular genes.
1. A method of selecting RYMV (Rice Yellow Mottle Virus) P1 proteins having PTGS (Post Transcriptional Gene Silencing) suppressor activity, said method comprising:
cloning genes encoding RYMV P1 proteins downstream of a RYMV 35S promoter to form expression cassettes, said expression cassettes containing a GUS reporter gene,
inoculating the expression cassettes encoding the suppressor proteins by biolistics onto rice leaves,
mechanically inoculating RYMV virus into said rice leaves,
assaying the viral load by DAS-ELISA using a polyclonal antibody directed against the virus,
visualizing the restoration of expression of the GUS reporter gene encoding beta-glucoronidase by histochemical assay,
quantifying the restoration of expression of the GUS reporter gene by assaying the enzymatic activity of GUS,
detecting siRNAs as evidence of PTGS by Northern blotting, and
selecting P1 proteins having PTGS suppressor activity.
2. The method of claim 1 , wherein the RYMV P1 protein is selected from a Tz3 RYMV isolate, a Tz8 RYMV isolate, an Mg1 RYMV isolate, and an CI63 RYMV isolate.
3. The method of claim 2 , wherein the RYMV P1 protein is a CI63 RYMV isolate or a Tz3 RYMV isolate.
4. The method of claim 2 , wherein the Tz8 P1 protein is selected for an intermediate level of effectiveness for PTGS suppression, and the Mg1 P1 protein is selected for a low level of effectiveness in PTGS suppression.
5. A viral vector, comprising a gene encoding a RYMV P1 protein selected from a Tz3 RYMV isolate, a Tz8 RYMV isolate, an Mg1 RYMV isolate, and an CI63 RYMV isolate, wherein said vector is a virus induced gene silencing (VIGS) vector and contains an insert of less than 50 bp in size, said insert being inserted in said vector in sense or antisense orientation, the target of said insert being a host mRNA transcript.
6. A viral vector, comprising a gene encoding a RYMV P1 protein selected from a Tz3 RYMV isolate, a Tz8 RYMV isolate, an Mg1 RYMV isolate, and an CI63 RYMV isolate, wherein said vector is a viral protein expression vector and contains an insert of greater than 700 bp of a gene of interest to be overexpressed, said insert being inserted into the replicative ORF 2a and 2b of RYMV.