Calcineurin

Antifungal susceptibility was determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol, as described [21] (link). Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of micafungin (MF, generously provided by Julia R. Köhler) or caspofungin (CS, generously provided by Rochelle Bagatell). Echinocandin gradients were typically from 2 µg/ml down to 0 with the following concentration steps in µg/ml: 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.00195313. For gradients from 16 µg/ml down to 0, the concentration steps in µg/ml were: 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼10³ cells were inoculated into each well. Geldanamycin (GdA, A.G. Scientific, Inc.) and radicicol (RAD, A.G. Scientific, Inc.) were used to inhibit Hsp90 at the indicated concentrations, and cyclosporin A (CsA, CalBiochem) and FK506 (A.G. Scientific, Inc.) were used to inhibit calcineurin at the indicated concentrations. Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of MF across the x-axis of the plate and 2-fold dilutions of either GdA or CsA across the y-axis of the plate. Plates were inoculated as with MIC tests. Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the vehicle for GdA, RAD, CsA, and FK506. Sterile water was the vehicle for MF and CS. Plates were incubated in the dark at 30°C for the time period indicated, at which point plates were sealed and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and was corrected for background from the corresponding medium. Each strain was tested in duplicate on at least two occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.3 (http://jtreeview.sourceforge.net).

Antifungal tolerance and resistance were determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol as described [25] (link), [27] (link). Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the solvent for fenpropimorph (FN, Sigma Aldrich Co) and terbinafine (TB, Sigma Aldrich Co.); fluconazole (FL, Sequoia Research Products) and micafungin (MF, generously provided by Julia R. Köhler) were dissolved in sterile ddH₂O. Geldanamycin (GdA, A.G. Scientific, Inc.) was used to inhibit Hsp90 at the indicated concentrations. Cyclosporin A (CsA, Calbiochem) was used to inhibit calcineurin at the indicated concentrations. Cercosporamide and staurosporine (STS, A.G. Scientific, Inc.) were used to inhibit protein kinase C at the indicated concentrations. DMSO was the solvent for GdA, CsA, STS, and cercosporamide.
Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of FL, FN, TB and cercosporamide. FL gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. FN gradients were from 25 µg/ml down to 0 with the following concentration steps in µg/ml: 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.04882813, 0.02441406. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.24414063. Cercosporamide gradients were from 100 µg/ml with the following concentration steps in µg/ml: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼10³ cells were inoculated into each well. Plates were incubated in the dark at 30°C or 35°C for the period of time indicated in the figure legend, at which point plates were sealed with tape and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and corrected for background from the corresponding medium. Each strain was tested in duplicate on at least 3 occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.1 (http://jtreeview.sourceforge.net).
Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of cyclosporin A across the x-axis of the plate and 2-fold dilutions of STS across the y-axis of the plate. STS gradients were from 0.5 µg/ml to 0 in the following concentrations steps in µg/ml: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125. CsA gradients were from 48 µg/ml down to 0 in the following concentration steps in µM: 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875. Plates were inoculated and growth was measured as with MIC tests. To test for synergy, the fractional inhibitory concentration (FIC) was calculated as follows: [(MIC₈₀ of drug A in combination)/(MIC₈₀ of drug A alone)] + [(MIC₈₀ of drug B in combination)/(MIC₈₀ of drug B alone)]. Values of ≤0.5 indicate synergy, those of >0.5 but <2 indicate no interaction and those ≥2 indicate antagonism.

CN activity in vitro was determined according to the instructions of recombinant/purified CN activity kits (Abcam). Briefly, after calcineurin and calcineurin assay buffer were mixed, different concentrations of tutin are added for 5 min. The calcineurin substrate was added to start the reaction. This assay was then terminated by thegreen assay reagent (100 μL). The absorbance of OD₆₂₀ was detected and the CN activity was calculated.

The leaf epidermis tissue (100 mg) was homogenized with liquid nitrogen, and total RNA was extracted using Trizol reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized following the instructions of the HiScript III RT SuperMix for qPCR (Vazyme). Real-time quantitative PCR was performed using ChamQ SYBR qPCR Master mix (Vazyme) in a Bio-Rad iCycler iQ5 fluorescence real-time PCR system (Bio-Rad, Hercules, CA, USA). The annealing temperatures of the target genes were all optimized to 58°C. The primers for quick-activating anion channel 1 (QUAC1), slow-activating anion channel (SLAC1), malate transport ATPase (ABCB14), potassium ion inflow channel (KAT1), potassium ion efflux channel (KOR1), calcineurin binding protein (CBL1), calmodulin kinase (CIPK23) are shown in Table S1. To normalize results, the Cq (quantification cycle value) was calculated as the relative expression level of each target gene and the housekeeping gene (Actin) in each sample.