OsCASP1 forms complexes with itself and OsCASP2 in rice

ABSTRACT

OsCASP1 (Casparian strip domain protein 1) was recently identified to function in Casparian strip (CS) formation at the endodermal cells in rice roots, which was required for selective mineral uptake in rice. Here, we further investigate the functional form of OsCASP1 in vivo. Expression analysis shows that OsCASP1, OsCASP2, OsCASP3, and OsCASP5 were expressed in roots apart from OsCASP4. A yeast two-hybrid (Y2H) assay revealed that OsCASP1 can interact with itself and OsCASP2, but not with OsCASP3 and OsCASP5. These interactions of OsCASP1 with itself and OsCASP2 at the plasma membrane were confirmed using bimolecular fluorescence complementation (BiFC) in rice protoplasts. These results indicated that OsCASP1 can form complexes with itself and OsCASP2 in rice roots.

The plant root is a specialized organ that selectively acquires water and nutrients from the soil environment. The endodermis, forming a major extracellular diffusion barrier in the root, helps to establish root selectivity in nutrient uptake. This diffusion barrier embedded in the anticlinal cell wall between endodermal cells is called the Casparian strip (CS).¹ Therefore, the CS is an important barrier regulating the selective entry of water and nutrients into root tissue. In Arabidopsis, several key genes involved in CS formation have been identified such as CASP1, MYB36, PER64 and ESB1.^2-6 Mutation of these genes resulted in defects of CS formation at the endodermis in roots and complex changes of the shoot ionome.⁷ In contrast with the situation in Arabidopsis, little is known about the molecular mechanism of CS formation in rice.

Different from Arabidopsis roots, there is one CS at both the exodermis and the endodermis in rice roots.⁸ Recently, OsCASP1 was reported to be only involved in CS formation at the endodermis in rice roots, which played an important role in regulating root selective uptake of some mineral elements especially Ca and Si.⁹ In this report, we further investigate the functional form of OsCASP1 in vivo. In Arabidopsis, AtCASPs have been shown to reside at the Casparian strip membrane domain (CSD) in heteromultimer with each other.² RT-PCR analysis showed that the expression of OsCASP1, OsCASP2, OsCASP3, and OsCASP5 was detected in rice roots except for OsCASP4 (Figure 1), which was consistent with previous result.⁹ Transient co-expression of OsCASP1-GFP, OsCASP2-GFP, OsCASP3-GFP, or OsCASP5-GFP with plasma membrane-localized marker mCherry-OsRac3¹⁰ in rice protoplast cells showed that the green fluorescence of four CASP-GFP fusion proteins was mainly merged with the mCherry-OsRac3 signal on the plasma membrane (Figure 2), indicating that they are plasma membrane-localized proteins. To determine whether OsCASP1 can directly interact with itself and other OsCASP proteins expressed in roots, we first used split-ubiquitin Y2H assays, where OsCASP1 was fused to the C-terminal half of the ubiquitin gene (Cub), and OsCASP1, OsCASP2, OsCASP3, or OsCASP5 was fused to the mutated N-terminal half of the ubiquitin gene (NubG). When co-transformed with OsCASP1-Cub and NubG-OsCASP1, or with OsCASP1-Cub and NubG-OsCASP2, yeast cells were able to grow on the selective medium, indicating that OsCASP1 can interact with itself and OsCASP2 (Figure 3). By contrast, when yeast cells were co-transformed with OsCASP1-Cub and NubG-OsCASP3, or with OsCASP1-Cub and NubG-OsCASP5, yeast growth was significantly inhibited as the negative control (Figure 3), suggesting that OsCASP1 cannot interact with OsCASP3 or OsCASP5.

Figure 1.

Expression analysis of five OsCASP genes in rice roots. The roots of 15-day-old wild-type seedlings grown in a nutrient solution were sampled for RNA extraction. Histone H3 was used as an internal standard.

Figure 2.

Subcellular localization of OsCASP1, OsCASP2, OsCASP3 and OsCASP5. Four OsCASP-GFP fusion proteins were co-expressed with mCherry-OsRac3 for marking plasma membrane in rice protoplasts, respectively. GFP (green), mCherry (red), and merged image of GFP (green) and mCherry (red) were shown. Scale bars, 10 μm.

Figure 3.

Identification of the interaction of OsCASP1 with itself and OsCASP2 by DUALmembrane system. OsCASP1 was fused to the C-terminal half (Cub). OsCASP1, OsCASP2, OsCASP3, and OsCASP5 were fused to the mutated N-terminal half (NubG) of ubiquitin. Yeast cells were co-transformed with various plasmid combinations as indicated at the right and were grown on SD medium without Leu and Trp as a control (left panel) or on a selective medium for protein interaction (right panel) without Leu, Trp, His, and Ade at different concentrations (10 times dilution from left side). All plates were grown for 3 d at 30°C.

To confirm that OsCASP1 can interact with itself and OsCASP2, but not with OsCASP3 and OsCASP5 in plant cells, bimolecular fluorescence complementation (BiFC) assays were performed using rice protoplast cells. As the negative controls, co-expression of OsCASP1-nYFP and the empty cYFP vector as well as nYFP and OsCASP2-cYFP did not enable the cells to produce the YFP signal (Figure 4). Similar to the negative control, the YFP signal was not observed in the cells co-expressing OsCASP1-nYFP with OsCASP3-cYFP or OsCASP5-cYFP (Figure 4), indicating that OsCASP1 cannot interact with OsCASP3 or OsCASP5 in plant cells. In contrast, in rice protoplasts co-expressing OsCASP1-nYFP with OsCASP1-cYFP or OsCASP2-cYFP, YFP signal could be restored and mainly co-localized with the mCherry signal of the plasma membrane marker (Figure 4), OsRac3, confirming in vivo interaction of OsCASP1-OsCASP1 and OsCASP1-OsCASP2 at the plasma membrane. Furthermore, some strong dot-like signals were observed at the plasma membrane, suggesting that interactions of OsCASP1-OsCASP1 and OsCASP1-OsCASP2 are likely to form homomultimer with itself or heteromultimer with OsCASP2 in vivo. Given that the expression level of OsCASP1 in the roots was more than 10-fold higher than that of OsCASP2,⁹ homomultimer of OsCASP1 by interaction with itself may be main form localized at the CSD between endodermal cells.

Figure 4.

BiFC visualization of the interaction of OsCASP1 with itself and OsCASP2 in rice protoplasts. Rice protoplasts were co-transformed with three constructs that expressed, respectively, mCherry-OsRac3 for marking plasma membrane, OsCASP1-nYFP and OsCASP1-cYFP, OsCASP2-cYFP, OsCASP3-cYFP, or OsCASP5-cYFP. Pair of plasmid constructs (negative control) with mCherry-OsRac3 were also transiently introduced into rice protoplasts. YFP (green), mCherry (red), and merged image of YFP (green) and mCherry (red) were shown. Scale bars, 10 μm.

In conclusion, our results demonstrated that OsCASP1 can interact with itself and OsCASP2 at the plasma membrane, and form complexes in rice.

Funding Statement

This work was supported by the Natural Science Foundation [2016GXNSFFA380013].