Mixture assay contained 10 mM d-galactose, 100 mM Tris-HCl buffer (pH 8.6), 2.5 mM NAD+, and the enzyme solution in a total volume of 1 mL.
The standard assay contained 40 mM potassium phosphate buffer (pH 7.5), 2 mM 3HB-CoA, 10 mM 5,5'-dithio-bis 2-nitrobenzoic acid) and 35,5'-dithio-bis 2-nitrobenzoicuble protein fraction (Bhubalan et al. [2011]).
Reaction mixtures for the standard assay contained in a total volume of 2.0 mL: 50 mM phosphate buffer pH 7.4, 1 mM NADH, 0.25 mM dye solution, and 200 μL of enzyme solution.
The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control.
To evaluate the glutamine synthetase (GS EC 6.3.1.2) activity, the mixture for the assay contained 90 mM imidazole-HCl (pH 7.0), 60 mM hydroxylamine (neutralized), 20 mM Na2KAsO4, 3 mM MnCl2, 0.4 mM ADP, 120 mM glutamine and the appropriate amount of enzyme extract.
NADH-dependent glutamate synthase (NADH-GOGAT EC 1.4.1.14) assay contained 25 mM Hepes-NaOH (pH 7.5), 2 mM L−1 glutamine, 1 mM α-ketoglutaric acid, 0.1 mM β-NADH, 1 mM Na2 EDTA and 100 μL of enzyme extract in a final volume of 1.1 mL.
Each assay contained a duplicate and a minus RT control.
Briefly, each assay contained 100 mM bicine, pH 7.8; 0.42 mM MTT, and 1.66 mM PMS.
Each assay contained two different Taqman probes, uniquely labeled to bind to separate (major versus minor) alleles.
MeDH activity was measured as described [1] except the assay contained 0.4 mM KCN. Ccr activity was measured as described [44] except that 2 mM NADPH was used.
However, the lowest dilution detected with the TaqMan®-based assay contained only 8×102 copies of ONNV cDNA/reaction (Fig. 3; B and D).
