Plant transactivation interaction motifs and uses thereof

Inventors

Petolino, Joseph F. • Li, Jianquan • Evans, Steven L. • Blue, Ryan C.

Assignees

Corteva Agriscience LLC

Abstract

This disclosure concerns compositions and methods for increasing the expression of a polynucleotide of interest. Some embodiments concern novel transactivation polypeptides and variants thereof that have been identified in plants, and methods of using the same. Particular embodiments concern the use of at least one DNA-binding polypeptide in a fusion protein to target at least one transactivation polypeptide or variant thereof to a specific binding site on a nucleic acid comprising the polynucleotide of interest, such that its expression may be increased.

Core Innovation

A plant-based transcriptional activation system is described that uses plant transactivation domains and derived transactivation domain interaction motifs. A plant-derived transactivation domain interaction motif, including sequence-specified motifs and engineered conservative variants, is incorporated into synthetic transcriptional activator fusion proteins. The system combines a plant transactivation interaction motif with heterologous sequence-specific DNA-binding domains to regulate transcription.

The synthetic transcriptional activator fusion proteins comprise a plant-derived transactivation domain interaction motif fused to a heterologous, sequence-specific DNA-binding domain. The DNA-binding domain context includes examples such as ZFP/Z6, GAL4, TAL/UPA, and UPA-box. The transactivation domain interaction motif is based on the interaction motif peptide of SEQ ID NO:22 and includes variants.

The disclosure emphasizes regulation effects that differ from VP16. Some motifs and variants enhance transcription more than VP16, while other motifs yield lower or no activity in assays. Yeast MEL1 reporter testing and plant tobacco (Nicotiana tabacum) reporter assays (gus) are used to compare native versus variant motifs and different DNA-binding domain contexts, with reported outcomes supporting the unexpected regulation behavior.

Claims Coverage

Two independent claims are identified. Both claims cover synthetic transcriptional activator fusion proteins defined by the same general architecture: a single DNA-binding peptide combined with a heterologous transactivation domain that includes an interaction motif peptide of SEQ ID NO:22 and specific sequence constraints for the transactivation domain.

Overall, the claims are grounded in the same core construct: a fusion protein that pairs a single DNA-binding peptide with a heterologous transactivation domain built from the interaction motif peptide of SEQ ID NO:22, where the transactivation domain is constrained to SEQ ID NO:109/SEQ ID NO:110 either by inclusion of exactly those sequences or by an amino acid identity threshold.

Stated Advantages

Documented Applications