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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 - New Phytol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China; MOA Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing, 210095, China.

No MeSH data available.


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).
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fig03: 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).

Mentions: We constitutively expressed the fusion protein OsNAR2.1 and GFP using the pSAT6-EGFP-C1 and pSAT6A-EGFP-N1 expression vector (Tzfira et al., 2005) to investigate the subcellular localization of OsNAR2.1. We transfected OsNAR2.1-GFP and GFP-OsNAR2.1 fusions into rice blade protoplasts under control of the cauliflower mosaic virus 35S promoter, and GFP expression was determined using confocal microscopy. To further explore the localization of OsNAR2.1, we expressed the OsNAR2.1-GFP and GFP-OsNAR2.1 constructs in tobacco epidermis cells (Supporting Information Fig. S3). GFP signaling demonstrated that the fusion protein was transiently expressed in both PM and cytoplasm whether the GFP was fused to the N- or C-terminus of OsNAR2.1 (Fig. 3a–d, Supporting Information Fig. S3).


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 - New Phytol. (2014)

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).
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fig03: 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).
Mentions: We constitutively expressed the fusion protein OsNAR2.1 and GFP using the pSAT6-EGFP-C1 and pSAT6A-EGFP-N1 expression vector (Tzfira et al., 2005) to investigate the subcellular localization of OsNAR2.1. We transfected OsNAR2.1-GFP and GFP-OsNAR2.1 fusions into rice blade protoplasts under control of the cauliflower mosaic virus 35S promoter, and GFP expression was determined using confocal microscopy. To further explore the localization of OsNAR2.1, we expressed the OsNAR2.1-GFP and GFP-OsNAR2.1 constructs in tobacco epidermis cells (Supporting Information Fig. S3). GFP signaling demonstrated that the fusion protein was transiently expressed in both PM and cytoplasm whether the GFP was fused to the N- or C-terminus of OsNAR2.1 (Fig. 3a–d, Supporting Information Fig. S3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China; MOA Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing, 210095, China.

No MeSH data available.