The University of Texas System

Ivan L Ingelbrecht

T Erik Mirkov

The present disclosure includes methods of using viral and plant proteins to regulate silencing in plants such as monocots. Some embodiments include methods of increasing expression of transgenes in plants. Such methods may include selecting a protein involved in post-translational gene silencing (PTGS), for e.g., a protein active in PTGS or a suppressor of PTGS, in a plant and transforming the plant with a nucleic acid encoding the protein involved in PTGS wherein expression of the protein involved in PTGS increases expression of the transgene in the plant. One non-limiting example of such a protein is the Sorghum Mosaic Virus (SrMV) P1/HC-Pro. Methods of the disclosure may be used to regulate silencing of transgenes and to increase expression of transgenes in a variety of plants such as sugarcane, corn, sorghum, and/or rice.

Isolation of proteins involved in posttranscriptional gene silencing and methods of use

The present invention includes a method for detecting and isolating sugarcane proteins that interact with the HC-Pro and P1 proteins of SrMV and other proteins involved in gene silencing, particularly in sugarcane. The method uses a two hybrid assay with an HC-Pro, P1, or other silencing-related protein-containing bait protein and a prey protein containing a polypeptide encoded by a DNA molecule in a cDNA library. The method also includes identification of false positives through reverse two-hybrid assays and using in vitro techniques such as farwestern blots or pull down assays where plant physiological conditions may be replicated. Finally, interactions may be confirmed in planta. Some novel proteins used in and discovered using the these methods are also identified. Methods of using viral and plant proteins to regulate silencing in plants such as sugarcane are also discussed.

The present invention discloses transgenic plant cells transformed with a nucleic acid encoding a SrMV P1/HC-Pro protein, wherein expression of the protein suppresses post-transcriptional gene silencing in the cell. The transgenic plant cell may be located in an embryonic callus or in a mature plant. The plant may be a monocot plant.

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Jabbeke, Belgium

Ivan L Ingelbrecht