X gluc

GUS staining was performed essentially as described [39 ]. Apical leaves of transformed tobacco plants (or tissue samples), one month after sub culture were incubated overnight at 37°C in a solution containing 1 mM X-Gluc (Fermentas, USA), 50 mM sodium phosphate buffer pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 1 mM Na₂EDTA, 0.1% Triton X100. The chlorophyll was removed using 70% ethanol. Petioles and leaves were sectioned by hand after staining. The samples were observed and photographed under Nikon 80I microscope (Nikon, Japan).

Flowering plants were assessed for GUS activity in leaf, spikelet, stigma, and anther tissue. The GUS activity was determined by incubating tissue samples in GUS assay solution (50 mM NaCl, 1 mM Tris, 1 mg·L⁻¹ X-Gluc (Thermo Fisher Scientific, Waltham, MA, USA) and 0.1% (v/v) Triton X-100) for 12 to 16 h. The samples were then washed and de-stained multiple times with 70% (v/v) ethanol.

GUS assays were performed as described earlier [37 (link)]. Briefly, samples were collected in ice-cold 90% acetone, incubated at room temperature for 20 min, followed by washing with ice-cold staining buffer (50 mM sodium phosphate, pH 7.0, 0.2% Triton X-100, 5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide). For pUBQ10::miR319c-GUS, staining buffer containing 10 mM potassium ferrocyanide and 10 mM potassium ferricyanide was used. Fresh staining buffer was added with 2 mM X-Gluc (R0851, Thermo Scientific, USA) and incubated for 4–6 hours (for pTCP4::GUS; pTCP4::TCP4-GUS, pUBQ10::miR319c-GUS and pMIR319C::GUS^#2) or overnight (for pMIR319C::GUS^#1) at 37°C in dark. Staining buffer was replaced with 70% ethanol, and 70% ethanol was changed 3–4 times in regular intervals to clear the tissue. Shoot apices and leaf primordia were dissected and mounted on a glass slide containing lactic acid under the microscope and were imaged using Olympus BX51 trinocular DIC microscope fitted with ProgResC3 camera and ProgResCapture Pro2.6 software (Jenoptik, Germany) with the following settings, exposure- 65ms, brightness-10 units, contrast- 35 units, colour temperature- 5000K, Image resolution- (1040 x 770 2X Bin), while keeping all other settings in default mode.

All plasmids were
propagated in Escherichia coli strain
DH5α. Solid (2% agar) or liquid Luria broth (LB) medium supplemented
with 100 μg/mL ampicillin was used as growth medium.
Aspergilli
strains used in this study are listed in Supplementary Table S2. All strains were cultivated using liquid or solid
(2% agar) minimal medium (MM) (1% glucose, 1× nitrate salt solution,^{42 (link)} 0.001% Thiamine, 1× trace metal solution),^{43 (link)} which was supplemented with 10 mM uridine
(uri), 10 mM uracil (ura), and/or 4 mM l-arginine
(arg) when required. To perform blue/white screening, solid MM was
supplemented with 0.115 mM X-Gluc (Thermo Fisher Scientific). Transformation
medium (TM) was prepared as MM, except for glucose, which was substituted
with 1 M sucrose.

Two-week-old seedlings of the homozygous T4 generation were used for GUS histochemical staining using X-Gluc (5-bromo-4-chloro-3-indonyl β-D glucoronide; Thermo Scientific) as a substrate as described in (Jefferson et al., 1987 (link); Divol et al., 2013 (link)). The staining was performed on five independent, single-insertion, homozygous lines grown on B5 media, and representative lines are shown.