Figure 6.

Broad Adaptability and Versatility of the ETPamir Screen.

(A) Time-course ETPamir screens for optimal amiRNA silencing GFP (35S-driven) in Arabidopsis mesophyll protoplasts. The numerical order of each amiR-GFP was based on the high-to-low WMD ranking. Luciferase (LUC) expression served as an internal control. Ctrl, control.

(B) Preexpression of amiR-GFP-4 but not amiR-GFP-1 suppresses GFP expression. As shown by the flow diagram, expression of nuclear GFP (NLS-GFP) was induced by a 1-h heat shock pulse after 3 h of constitutive expression of amiR-GFPs in Arabidopsis protoplasts. Images of NLS-GFP with ∼300, 150, and 180 cells are shown from left to right.

(C) Rapid ETPamir screens in protoplasts from diverse plant species. Expression of GFP was induced by a 1-h heat shock pulse after 3 h of constitutive expression of amiR-GFPs. Heat shock–inducible LUC served as an internal control.

(D) Rapid ETPamir screens in rice protoplasts. Expression of GFP was induced by a 1-h heat shock pulse after 3 h of constitutive expression of amiR-GFPs. Unlike the amiR-GFPs in (A) to (C) that were derived from Arabidopsis miR319a (ath-miR319a), amiR-GFPs derived from rice miR528 (osa-miR528) were used.

(E) Sequence alignment between miR319a and putative target sites. Mismatches in miR319a or miR319a¹²⁹ (nonfunctional variant) to individual predicted target sequences are highlighted in red.

(F) Validation of predicted target genes for miR319a using protein-based miRNA target screens. Expression of target candidates was induced by a 1-h heat shock pulse after 3 h of constitutive expression of miR319a or miR319a¹²⁹. TCP20, as a known untargeted gene, was tested as a negative control to establish the physiological specificity of the screen. Heat shock–inducible GFP-HA served as an internal control.

All experiments were repeated at least three times with similar results.