In Vivo Monitoring of Cytosolic pH Using the Ratiometric pH Sensor pHluorin

Fusarium oxysporum Casein Kinase 1, a Negative Regulator of the Plasma Membrane H+-ATPase Pma1, Is Required for Development and Pathogenicity

“…The H + -ATPase activity of Pma1 in F. oxysporum was measured in the plasma membrane fraction of germlings as previously described [35] with minor modifications [36] by reading absorbance at 750 nm in a spectrofluorometer (Infinite M200 PRO, TECAN Life Sciences, 8708 Männedorf, Switzerland). Specific Pma1 H + -ATPase activity was calculated by subtracting the residual activity obtained after the addition of the specific Pma1 inhibitor DES, from the total activity (addition of methanol), expressed in mmol/min/g protein and normalized to time zero.…”

“…Measurements of pH c in F. oxysporum germlings were performed, as previously described [36], by reading fluorescence emission at 510 nm after excitation at 395 nm and 475 nm in a TECAN spectrofluorometer (Infinite M200 PRO, TECAN Life Sciences, Männedorf, Switzerland). The values of the pHluorin-negative wt background were subtracted for each wavelength, and the 395/475 nm ratio was calculated and converted to pH c values using a pH calibration curve [36]. Experiments were performed at least twice with three independent replicate wells each.…”

“…Western blot analysis of MAPK phosphorylation levels was performed as described [36,38,39]. Phosphorylated Mpk1 and Fmk1 was detected using rabbit antiPhospho-p44/42 MAPK (Erk1/2) antibody (Thr202/Tyr204; Cell Signaling Technology; #4370), while total Mpk1 and Fmk1 protein was detected with mouse monoclonal anti-Mpk1 antibody (Santa Cruz Biotechnology; sc165979) and a custom-designed polyclonal anti-Fus3 antibody [36]. Mouse anti-α-tubulin antibody (Sigma-Aldrich; #T9026) was used as the loading control.…”

“…To study how ck1 deletion affects pH c , we introduced the gene knockout construct, with the phleomycin resistance cassette, into a wt background expressing the fluorescent ratiometric pH c sensor pHluorin [36]. PCR analysis identified six phleomycin-resistant transformants producing amplification patterns indicative of a homologous replacement of the ck1 gene.…”

“…Southern blot analysis of genomic DNA confirmed the replacement of a 4.1 kb NsiI hybridizing fragment corresponding to the wt ck1 allele, by a hybridizing fragment of 2.5 kb, consistent with homologous insertion of the deletion construct at the ck1 locus (Figure S2C). We next used ratiometric pHluorin-based measurements [36] to compare pH c in the wt and the ck1∆ mutant. We found that the homeostatic pH c of the ck1∆ mutant under the conditions studied was around 6.1, which is approximately 0.7 pH units lower than the pH c of the wt strain (Figure 4C).…”