Direct and tunable modulation of protein levels in rice and wheat with a synthetic small molecule

Abstract

Direct control of protein level enables rapid and efficient analyses of gene functions in crops. Previously, we developed the RDDK-Shield1 (Shld1) system in the model plant Arabidopsis thaliana for direct modulation of protein stabilization using a synthetic small molecule. However, it was unclear whether this system is applicable to economically important crops. In this study, we show that the RDDK-Shld1 system enables rapid and tunable control of protein levels in rice and wheat. Accumulation of RDDK fusion proteins can be reversibly and spatio-temporally controlled by the synthetic small-molecule Shld1. Moreover, RDDK-Bar and RDDK-Pid3 fusions confer herbicide and rice blast resistance, respectively, in a Shld1-dependent manner. Therefore, the RDDK-Shld1 system provides a reversible and tunable technique for controlling protein functions and conditional expression of transgenes in crops.

Keywords: RDDK-Shld1 system; protein stability; rice; small molecule; wheat.

Figure 1

Shld1‐dependent accumulation of RDDK‐EGFP in rice. (a) Schematic of the RDDK–Shld1 system. Ub‐RDDK fusion gene is driven by maize ubiquitin promoter. During translation, ubiquitin fusion (Ub) is rapidly cleaved by endogenous deubiquitinating enzymes (DUBs) exposing the N‐terminal arginine (R). A lysine (K) is included as a potential recipient for ubiquitination of the fusion protein just after the destabilizing domain (DD). RDDK‐POI fusion proteins will thus be degraded by the 26S proteasome. Shld1 binds specifically to the DD domain such that the RDDK‐POI is stabilized. Ubi, maize ubiquitin promoter; POI, protein of interest; OCS, octopine synthase terminator; R, arginine; K, lysine. (b) Quantitative RT‐PCR analysis of RDDK‐EGFP fusion gene expression. Wild‐type (WT) and RDDK‐EGFP transgenic rice plants were treated with 3 μm Shld1 or mock treated for 8 h. Results were normalized to rice UBIQUITIN 5 (Os UBQ5), and expression level of the transgene in mock‐treated RDDK‐EGFP transgenic rice plants was set at one unit. Error bars indicate SD. (c) Immunoblotting with anti‐GFP antibody detects Shld1‐induced accumulation of RDDK‐EGFP in the transgenic rice plants. Plants were treated with 3 μm Shld1 or mock solution for 8 h. ACTIN was used as a protein loading control. (d) Shld1 dose‐dependent accumulation of RDDK‐EGFP detected by immunoblotting with anti‐GFP antibody. RDDK‐EGFP plants were treated with varying concentrations of Shld1 for 8 h. (e) Confocal images of epidermal cells of leaf sheaths from wild‐type (WT) and RDDK–EGFP plants treated with varying concentrations of Shld1 for 8 h. Bars = 50 μm. (f) Fluorescence intensity quantification of the confocal microscopy images in (e). Error bars indicate SD. a.u., arbitrary unit.

Figure 2

Temporal and spatial control of RDDK‐EGFP accumulation by Shld1 in rice. (a) Shld1‐induced RDDK‐EGFP accumulation is reversible in rice. 14‐day‐old rice plants were first treated with 3 μm Shld1 and washed 3 h later with water to remove Shld1. Leaves were collected at the indicated times of Shld1 application with ‘0’ as leaves collected just before Shld1 application. Levels of RDDK‐EGFP were detected by immunoblotting with anti‐GFP antibody. (b) A representative picture showing 3 μm Shld1‐treated local leaf and systemic leaf without Shld1 treatment on a rice seedling. (c) Immunoblotting with anti‐GFP antibody of total protein extracts from local and systemic leaves as in (b).

Figure 3

Shld1‐induced herbicide resistance in RDDK‐Bar transgenic rice plants. Shld1‐induced accumulation of RDDK‐Bar fusion protein in rice. Immunoblotting with anti‐HA antibody of total extracts from WT and RDDK‐Bar transgenic rice plants treated with or without 10 μm Shld1 for 8 h. ACTIN was used as a protein loading control. (b) Shld1‐induced bialaphos resistance of RDDK‐Bar plants as shown by the CR assay. Leaf pieces of WT and RDDK‐Bar plants were excised and cultured separately in a 24‐well plate with CR medium supplemented with (‘+’) or without (‘−’) 8 mg/L bialaphos and/or 10 μm Shld1. The plate was incubated in a growth chamber at 24 °C with a light/dark cycle of 16 h/8 h. Photographs were taken 3 days after treatment. (c) Basta resistance of rice plants induced by Shld1. 14‐day‐old WT and RDDK‐Bar plants were first treated with or without 10 μm Shld1 for 3 h before spraying once with Basta. Photographs were taken 10 days after treatment. (d) Shld1 conferred spatial control of herbicide resistance in RDDK‐Bar transgenic rice plants. Local leaf of a 14‐day‐old RDDK‐Bar plant was treated with 10 μm Shld1; 3 h later, both local and systemic leaves were sprayed with Basta. Shld1‐treated WT plants and mock‐treated RDDK‐Bar plants were sprayed with Basta as controls. Photographs were taken 10 days after treatment.

Figure 4

Shld1‐induced race‐specific resistance of rice to the blast fungus Magnaporthe oryzae. Shld1‐dependent resistance of RDDK‐Pid3 plants to an avirulent strain of M. oryzae. Leaf sheaths were detached from 4‐week‐old rice plants and inoculated with a suspension of 2 × 10⁵ mL⁻¹ fungal spores of M. oryzae strain Zhong‐10‐8‐14 with 10 μm Shld1 (indicated as ‘Shld1’) or without Shld1 (indicated as ‘Mock’). Shown are bright‐field images of sheath cells 48 h postinfection stained with Trypan blue to highlight the fungus and host cell death. Bars = 25 μm. (b) Quantitative analysis of compatible and incompatible interactions of the rice sheath cells in the same experiment as in (a). (c) Shld1‐treated RDDK‐Pid3 plants remain susceptibility to virulent M. oryzae strain. Shown are confocal images of sheath cells 48 h postinfection with a GFP‐tagged M. oryzae strain ZB15. Representative images are shown. Bars = 25 μm.

Figure 5

Modulation of protein levels and function with the RDDK‐Shld1 system in wheat. Shld1‐induced accumulation of RDDK‐GUS fusion protein in wheat. Immunoblotting with anti‐HA antibody of total extracts from RDDK‐GUS transgenic and WT wheat plants. Plants were treated with 10 μm Shld1 or mock solution for 8 h. (b) GUS staining of leaf pieces of RDDK‐GUS and WT plants treated with 10 μm Shld1 or mock solution for 8 h. A representative image is shown. (c) Shld1‐induced accumulation of RDDK‐Bar fusion protein in wheat. Immunoblotting with anti‐HA antibody of total extracts from RDDK‐Bar and WT plants treated with 10 μm Shld1 or mock solution for 8 h. (d) Shld1‐induced bialaphos resistance of RDDK‐Bar transgenic wheat as shown by CR assay. Leaf pieces of WT and RDDK‐Bar transgenic wheat plants were excised and cultured separately in a 24‐well plate with CR medium supplemented with (‘+’) or without (‘−’) 8 mg/L bialaphos and/or 10 μm Shld1. The plate was incubated in a growth chamber at 24 °C with a light/dark cycle of 16 h/8 h. Photographs were taken 3 days after treatment.