Materials and reagents

Biological materials

1. Yeast strain: protease-deficient yeast strain BJ5465 (MATa, ura3–52, trp1, leu2Δ1, his3Δ200, pep4::HIS3, prb1Δ1.6R, can1, GAL+) (ATCC, catalog number: 208289TM), which was obtained from the Yeast Genetic Stock Center (University of California, Berkeley, CA).

2. Sensors: NIT domain/NasR

   The full-length open reading frame of NasR from Klebsiella oxytoca (Boudes et al., 2012) in the pDONR221 GATEWAY Entry vector was used as a sensory domain to create the nitrate sensors NiMet3.0 and nlsNiMet3.0. Note: Constructs were inserted using an Entry clone by Gateway LR reactions into the yeast expression vectors pDRFlip30.

   The NiMet3.0 is fused to the full-length of NasR with pDRFlip30 vector and sandwiched between an N-terminal Aphrodite t9 (AFPt9) variant (Deuschle et al., 2006), with nine amino acids truncated off the C terminus, and a C-terminal monomeric Cerulean (mCer) (Rizzo et al., 2006).

   Note: The pDRFlip30 vector was modified from pDRFlip39 vector (Addgene, catalog number: 65517).

3. Agrobacterium strain: Agrobacterium tumefaciens strain GV3101 was used here to obtain high transformation rates and high levels of expression, typically leading to high copy insertion into the genome (Koncz and Schell, 1986).

4. Arabidopsis thaliana: wild type Col-0, a nitrate transporter mutant [npf6.3/NTR1;1/chl1-5 (Leran et al., 2014)], and a nitrate reductase mutant [nia1nia2 (Desikan et al., 2002)] were used.

   
   

Reagents

1. Potassium nitrate (KNO₃) (Sigma-Aldrich, catalog number: P8394)

2. Potassium chlorate (KClO₃) (STREM, catalog number: 93-1913)

3. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405)

4. Potassium nitrite (KNO₂) (ARO, catalog number: 42306)

5. Potassium sulfate (K₂SO₄) (SHOWA, catalog number: 1648-4150-000-23)

6. Potassium sulfite (K₂SO₃) (ACROS, catalog number: 44021)

7. Potassium selenite (K₂SeO₃) (STREM, catalog number: 931971-000000-18)

8. Potassium molybdenum oxide, anhydrous (K₂MoO₄₎ (Alfa, catalog number: 22898-0000000-17)

9. Ammonium chloride (NH₄Cl) (Merck, catalog number: 1.01145-0500)

10. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M9272)

11. Gly-Gly (Sigma-Aldrich, catalog number: G1002)

12. YNB, yeast nitrogen base w/o amino acids w/o ammonium sulfate (BD, Difco, catalog number: 233520)

13. DO supplement-Ura (Takara Bio Company, Clontech, catalog number: 630416)

14. D-(+)-Glucose monohydrate (Fluka Analytical, catalog number: 49159)

15. Sucrose (Merck, catalog number: 1.07687.1000)

16. Agar (BD Bacto^TM, catalog number: DIF214530)

17. Agar (Phytoagar^TM, catalog number: 40100072)

18. MES hydrate (Sigma-Aldrich, catalog number: M2933)

19. MOPS (Sigma-Aldrich, catalog number: M3183)

20. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881)

21. 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: DTT-RO)

22. Carrier DNA [UltraPure^TM salmon sperm DNA solution (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 115632-011)]

23. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758)

24. Polyethylene glycol 4000 (PEG4000) (Fluka Analytical, catalog number: 81240)

25. Lithium acetate dihydrate (LiOAc) (Sigma-Aldrich, catalog number: L4158)

26. Tris hydrochloride ultrapure bioreagent (Tris-Cl) (J.T. Baker, catalog number: 4103-02)

27. Sodium chloride (NaCl) (Merck, catalog number: 1.06404.1000)

28. Sodium hydroxide (NaOH) (Merck, catalog number: 1.06498.1000)

29. Peptone (BD Bacto^TM, catalog number: 211677)

30. Yeast extract (BD Bacto^TM, catalog number: 212750)

31. MS modified basal salt mixture without nitrogen (MS) (PhytoTech Labs, catalog number: M531)

32. MilliQ or distilled water

33. Spectinomycin

34. Kanamycin

    
    

Solutions

1. 40% (w/v) glucose solution (sterile and filtrated)

2. MOPS buffer

3. MES buffer

4. Yeast extract peptone dextrose (YPD) medium (see Recipe 1)

5. Solid yeast nitrogen base (see Recipe 2)

6. PLATE mixture (the acronym of PEG, lithium acetate, Tris, and EDTA) (see Recipe 3)

7. Wash buffer (see Recipe 4)

8. Resuspension buffer (see Recipe 5)

9. Substrate addition (see Recipe 6)

10. Plant growth solid base (see Recipe 7)

    
    

Recipes

1. Yeast extract peptone dextrose (YPD) medium^{a, c}

   Reagent	Final concentration	Amount
   Peptone	2.0% (w/v)	10 g
   Yeast extract	1.0% (w/v)	5 g
   Agar	2.4% (w/v)	12 g
   40% sterile filtrated glucoseb	2% (w/v)	25 mL
   H2O	n/a	n/a
   Total	n/a	500 mL

   1. Autoclave, 121 °C, 15 psi, 15 min

   2. For liquid medium, when hand-warm, add glucose from 40% sterile filtrated stock to a final concentration of 2% under a sterile hood (e.g., biosafety cabinet).

   3. For solid medium, add 20 g/L agar before autoclaving. Add sterile filtrated glucose from 40% stock to a final concentration of 2% when the medium is hand-warm before pouring plates.

      
      

2. Solid yeast nitrogen base (-ura DropOut medium)^{a, c}

   Reagent	Final concentration	Amount
   Yeast nitrogen base w/o amino acids w/o ammonium sulfate	1.7 g/L	1.7 g
   DO supplement-Urad	0.77 g/L	0.77 g
   40% sterile filtrated glucoseb	2% (w/v)	50 mL
   H2O	n/a	to 1,000 mL
   Total	n/a	1,000 mL

   1. Autoclave, 121 °C, 15 psi, 15 min

   2. For liquid medium, when hand-warm, add glucose from 40% sterile filtrated stock to a final concentration of 2% under a sterile hood (e.g., biosafety cabinet).

   3. For solid medium, add 20 g/L agar before autoclaving. Add sterile filtrated glucose from 40% stock to a final concentration of 2% when the medium is hand-warm before pouring plates.

   4. Adjust the pH of the -ura DropOut medium to pH 5.8 with NaOH before addition of agar and autoclaving.

      
      

3. PLATE mixture for yeast transformation

   Reagent	Amount
   45% PEG4000	90 mL
   1 M LiOAc	10 mL
   1M Tris-Cl (pH7.5)	1 mL
   0.5M EDTA	0.2 mL
   Total	100 mL

   
   

4. Wash buffer

   Reagent	Final concentration	Amount
   MES	50 mM	9.76 g
   Total	n/a	1,000 mL

   1. Adjust the pH of the MES buffer to pH 5.5 with NaOH and autoclave.

   2. Autoclave, 121 °C, 15 psi, 15 min.

      
      

5. Resuspension buffer

   Reagent	Final concentration	Amount
   MES buffer (Recipe 4)	50 mM	250 mL
   Agar	0.05%	0.125 g
   Total	n/a	250 mL

   1. Wait until the medium cools to room temperature (RT) to delay sedimentation of the cells during the measurement.

      
      

6. Plant growth solid base^a–d

   Reagent	Final concentration	Amount
   MS salts without nitrogen	1/2 strength	0.78 g
   20% sterile filtrated sucrose	0.5 % (w/v)	25 mL
   Total	n/a	1,000 mL

   1. Adjust the pH to pH 5.5 with KOH before addition of agar and autoclaving.

   2. Autoclave, 121 °C, 15 psi, 15 min

   3. For liquid medium, when hand-warm, add sucrose from 20% sterile filtrated stock to a final concentration of 0.5% under a sterile hood (e.g., biosafety cabinet).

   4. For solid medium, add 12 g/L Phytoagar^TM before autoclaving. Add sterile filtrated glucose from 20% stock to a final concentration of 0.5% when the medium is hand-warm before pouring plates.

      
      

7. Substrate addition

   Reagent	Final concentration	Amount
   KNO3 or other substrates (Reagent 1–11)	Depending on the experimental design	-
   MES buffer	50 mM	-
   Total	n/a	50 mL

   Depending on the concentration of the nitrate needed, use the nitrate stock solution and dilute it with MES buffer or MOPS buffer.

   
   

Laboratory supplies

1. 96-well microplates (flat bottom clear or black) (Greiner Bio-One, catalog numbers: 650101 and 650209)

   Note: Black plates have a lower background.

2. Multichannel (12) pipette (for 100 μL) (e.g., Sartorius, catalog number: 725240)

3. 50 mL sterile plastic tubes (Falcon^®)

4. Petri dishes (diameter: 9 mm; height: 15 mm; sterile) (Alpha Plus, catalog number: BL6905)

5. Glass beads (3 mm) (BasicLife, catalog number: CEO-1169)

6. Vacuum-driven filter system (250 mL, upper cup, 0.22 μm PES) (AGC, catalog number: AGC-VC-PES22-250-1CS)

Equipment

1. Monochromator-based spectrofluorimeter for 96-well plates [Safire or Infinite^® M1000 (Tecan Trading)]

2. Cell density meter (Amersham, model: Ultrospec 10)

3. Orbital shaker, with temperature and velocity control (Eppendorf, New Brunswick Scientific, model: Innova 44)

4. Incubator for 28–30 °C incubation of yeast cells (YIHDER, model: LM-570RD)

5. Centrifuge with swinging rotor for 50 mL tubes (Eppendorf, model: 5810R)

6. Inverted confocal plus super resolution microscope (Zeiss, LSM 780 + ELYRA): high sensitivity confocal microscope is equipped with GaAsP spectrum detector, and the super resolution microscope is a Structure Illumination Microscopy.

7. The laboratory-established light-sheet system was made in cooperation with Microlambda Pte Ltd (Singapore)

8. Growth chamber

Software and datasets

1. MetaMorph software (Downingtown, PA)

2. Fiji (http://fiji.sc/)

3. GraphPad Prism version 9.0.0 for Mac (www.graphpad.com)

Procedure