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New Phytol
2014 Oct 01;2041:74-80. doi: 10.1111/nph.12986.
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Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport.
Liu X
,
Huang D
,
Tao J
,
Miller AJ
,
Fan X
,
Xu G
.
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A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.
Figure 1. Testing the interaction of OsNAR2.1 mutations with OsNRT2.3a using the DUAL membrane pairwise interaction kit with HIS3, ADE2 and lacZ as reporter genes. Yeast strain NMY51 carrying OsNRT2.3b (T2.3b) in the pPR3-N vector as prey, OsNAR2.1 (R) in the pBT3-C vector as bait and co-expression of T2.3b & R as the negative gene control for membrane protein interactions; and OsNRT2.3a (T2.3a) in the pPR3-N vector as prey, OsNAR2.1 (R) in the pBT3-C vector as bait and co-expression of T2.3a & R as a positive gene control for membrane protein interactions (Yan et al., 2011). (aâc) Cells grown on selective control SD-LW block (without Leu and Trp) medium or SD-AHLW block (without Ade, His, Leu and Trp); (dâf) β-galactosidase activity assay for quantification of the interaction strength. For a detailed description of each figure, for example in (a), SD-LW block rows 1â4 show T2.3b & R, T2.3a & R, T2.3a & R-R100G and T2.3a & R-D109N, respectively; in the SD-AHLW block, yeast growth was in the same order as in the SD-LW block. *Significant difference at P < 0.05 of the same treatments among the different combinations. The values represent the means ± standard deviation of five replicates.
Figure 2. Functional assay of OsNAR2.1 point mutations and OsNRT2.3a for nitrate transport in Xenopus oocytes. Uptake of 15NâNO3â into oocytes injected with water or mixtures, as indicated. Oocytes were incubated for 16 h in modified Barthâs saline containing 0.5 mM 15NâNO3â. Using water as a control; syn-T2.3a + syn-R: oocytes were injected with the mRNA mixture of codon-optimized and synthesized OsNAR2.1 (see Feng et al., 2013) and codon-optimized and synthesized OsNRT2.3a (see Feng et al., 2013); syn-T2.3a + ori-R: mRNA mixture of rice original OsNAR2.1 and codon-optimized and synthesized OsNRT2.3a; syn-T2.3a + ori-R-R100G: mRNA mixture of rice original OsNAR2.1 with the D109N mutation and codon-optimized and synthesized OsNRT2.3a; syn-T2.3a + ori-R-D109N: mRNA mixture of rice original OsNAR2.1 with the R100G mutation and optimized, synthesized OsNRT2.3a.
Figure 3. Localization of OsNAR2.1 in rice protoplasts. (a) FM4-64FX dye image; the red fluorescence reflects the position of the plasma membrane (PM). (b) Green fluorescent protein (GFP) fluorescence after expressing NAR2.1-GFP and GFP-NAR2.1 fusion proteins in rice blade protoplasts. (c) Rice protoplasts expressing FM4-64FX (red) and GFP (green) fluorescence. (d) Rice protoplasts in bright field without exciting light. Column 1 shows the protoplasts expressing 35S: GFP was used as a control. Column 2 shows the protoplasts expressing rice OsNAR2.1-GFP fusion protein with FM4-64FX dye. Column 3 shows the protoplasts expressing rice GFP-OsNAR2.1 fusion protein with FM4-64FX dye. FM4-64FX is a membrane-selective fluorescent vital dye. Bars, 10 μm. (e) Immunoblot for OsNRT2.3a, OsNAR2.1, PIP1 (PM marker), V-ATPase (vacuolar marker), and Bip (endoplasmic reticulum (ER) marker) in cell membranes separated from roots of two-month-old rice seedlings. Proteins from microsomes (M), PM and endomembranes (EM) were analyzed on 10% SDS-PAGE gels (50 μg of protein/lane).
Figure 4. Transient expression of split yellow fluorescent protein (YFP) constructs in rice protoplasts. (a) FM4-64FX dye image; red fluorescence reflects the position of the plasma membrane. (b) YFP fluorescence images: protoplasts were transfected with OsNRT2.3a-cEYFP (T2.3a) and nEYFP-OsNAR2.1 (R). (c) Rice protoplasts expressing EYFP (yellow) with FM4-64FX (red) fluorescence. (d) Rice protoplasts in bright field without exciting light. Column 1, protoplasts expressing YFP co-transfected with PSAT1-nEYFP-C1 and PSAT1-cEYFP-N1 as a control (eYFP). Column 2, protoplasts transfected with OsNRT2.3a-cEYFP (T2.3a) and nEYFP-OsNAR2.1 (R) with FM4-64 dye. Column 3, protoplasts transfected with OsNRT2.3a-cEYFP (T2.3a) and nEYFP-OsNAR2.1 R100G (R-R100G) with FM4-64FX dye. Column 4, protoplasts transfected with OsNRT2.3a-cEYFP (T2.3a) and nEYFP-OsNAR2.1 D109N (R-D109N) with FM4-64FX dye. Bars, 10 μm.
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