Fig 1.
Symptoms of potassium (K+) deficiency in banana plantlets infected with Fusarium oxysporum f. sp. cubense tropical Race 4 (Foc TR4) or treated with FSA.
(A) Comparison of symptoms between Fusarium wilt and K+-deficient banana plantlets. (B) Disease phenotype and (C) Disease index (DI) distribution in banana plantlets inoculated with Foc TR4 under varying K+ conditions. (D) Comparison of symptoms and (E) K+ content between FSA-treated and K+-deficient banana plantlets. Data are presented as means ± SE (n = 3). Student’s t-test, *p < 0.05; ***p < 0.001. (F) Effect of FSA on net K+ fluxes in the root tip zone of banana (indicated by the arrow).
Fig 2.
Electrical properties of MaAKT1 channels expressed in HEK-293 cells.
(A) Diagram illustrating whole-cell recording from HEK-293 cells overexpressing MaAKT1 channels. (B-C) Representative whole-cell current traces from control (pcDNA6 vector-transfected) and MaAKT1-transfected HEK-293 cells, recorded with the standard bath and pipette solutions. The insert shows voltage pulses from +6 mV to −194 mV in 20 mV decrements for 500 ms, with a holding potential of −34 mV. (C) Summarized current density of background current in HEK-293 cells (n = 9) and MaAKT1-mediated inward rectifying K+ current (n = 25). (D) Representative current traces recorded at −194 mV in the presence of 100 mM K+ (red), NH4+ (black), and Na+ (blue) in the bath solution. (E) Diagram illustrating inside-out recording from tiny patches excised from cell membrane of HEK-293 cells overexpressing MaAKT1 channels. (F) Representative single-channel current traces from inside-out patches excised from HEK-293 cells, showing inward currents at hyperpolarizing voltages. Pipette and bath solutions contained 150 mM and 100 mM K-gluconate, respectively (G) Single-channel conductance (γ) determined by linear regression (red solid line) (n = 4–11 for each data point).
Fig 3.
FSA inhibits MaAKT1 channels in a dose-dependent manner in HEK-293 cells.
(A) Time course of normalized currents at −194 mV in FSA-treated and control groups. The arrow indicates the application time of FSA or DMSO (n = 4). (B) Representative whole-cell current traces showing the response of MaAKT1 currents to various concentrations of FSA. (C) Summarized currents at −194 mV, showing a dose-dependent inhibition of MaAKT1 current by FSA, with an IC50 of 76.02 μM (n = 5–10 for each data point).
Fig 4.
FSA inhibits MaAKT1 channels by increasing intracellular ROS levels.
(A) Effect of 20 μM DPI (an RBOh inhibitor) on net K+ fluxes measured in epidermal roots cell of Col-0 root. (B) Net K+ fluxes measured from epidermal root cells of Col-0 and rbohd mutant root in response to 20 μM FSA. Data in (A) and (B) are presented as means ± SE (n = 10). (C and D) Representative DHE fluorescence images (C) and summarized DHE intensity (D) in control (left), FSA-treated (middle), and TEMPOL-pretreated, followed by FSA treatment (right) HEK-293 cells. Data are shown as means ± SE (n = 6). ***p<0.001 and ***p<0.0001 with one-way ANOVA followed by Newman-Keul’s test. (E and F) Representative whole-cell current traces (E) and summarized current density (F) of MaAKT1 current before and after FSA treatment in non-pretreated and TEMPOL-pretreated HEK-293 cells. Data are shown as means ± SE (n = 5). **p < 0.01 and ***p < 0.001 with one-way ANOVA followed by Newman-Keul’s test.
Fig 5.
S-glutathionylation mediates the oxidants-induced inhibition of MaAKT1 channels.
(A-D) The inhibition of MaAKT1 currents by thiol oxidants from the intracellular side. (A-C) Representative whole-cell current traces (top) and summarized current at −194 mV (bottom) showing MaAKT1 currents before and after application of the thiol oxidants, 2-DTP (A), DTBP (B), and DTNB (C). (D) Representative macroscopic current traces (top) and summarized current at −194 mV (bottom) showing changes in MaAKT1 currents following DTNB application. Data in (A) to (D) are shown as means ± SE (n = 5–6). Paired student’s t-test, ns denotes not significant, * p<0.05, **p < 0.01, and ***p < 0.001 compared with control. (E-H) GSH involvement in oxidant-induced inhibition of MaAKT1 channels. (E-G) Representative macroscopic current traces recorded in giant inside-out patches before and after application of H2O2 (E), diamide (F), with or without 300 μM GSH, and GSH alone (G). (H) Summary of the effects of H2O2, diamide, and GSH on MaAKT1 current. Data are shown as means ± SE (n = 4–7 for different treatments), *p < 0.05, ***p < 0.001 compared with control with one-way ANOVA followed by Newman-Keul’s test.
Fig 6.
FSA inhibits MaAKT1 channels through S-glutathionylation.
(A) DTT rescues MaAKT1 channels from inhibition by GSSG. (Left) Representative macroscopic current recorded in giant inside-out patches before (top), after 5 mM GSSG (middle), and after 5 mM GSSG followed by 5 mM DTT (bottom). (Right) Normalized current at −194 mV showing the effects of GSSG and DTT on MaAKT1 current. Data are shown as means ± SE (n = 6). One-way ANOVA followed by Newman-Keul’s test, **p < 0.01 and ***p < 0.001. (B) DTT in the patch pipette prevented MaAKT1 inhibition by FSA. Representative whole-cell current traces and summary of the preventive effect of DTT (5 mM) in the pipette on FSA-induced MaAKT1 inhibition (n = 3). Ns, not significant compared with control group. (C) FSA treatment decreases intracellular GSH level (left), increases GSSG level (middle), and reduces GSH/GSSG ratio (right) (n = 3). *p < 0.05 and **p < 0.01 compared with control. (D) GSH in the pipette prevents MaAKT1 inhibition by FSA. Representative whole-cell current traces and summary of the preventive effect of GSH (2 mM) in the pipette on FSA-induced MaAKT1 inhibition (n = 3). Ns, not significant compared with the control group. Data in (B) to (D) are shown as means ± SE and compared using Student’s t-test (C) or paired Student’s t-test (B and D). (E) Immunofluorescence showing that FSA treatment promotes the interaction between GSH (red) and MaAKT1 channels (green) (n = 5). Bar = 20 μm. (F) Co-IP results show that FSA treatment promotes the biochemical interaction between GSH and MaAKT1 channels (n = 3). (G) Representative whole-cell current recorded in HEK-293 cells overexpressing HA-tagged MaAKT1 channel.
Fig 7.
Cys202 is critical for FSA-induced S-glutathionylation of MaAKT1 Channels.
(A-D) Representative whole-cell current traces and summaries showing the effect of FSA on different MaAKT1 mutants, including (A) C202A, (B) C207A, (C) C215A, and (D) C218A. Data are shown as mean ± SE (n = 3–7 for each mutant). Ns, not significant, **p < 0.01, ***p < 0.001 compared with control using paired Student’s t-test. (E) Co-IP results indicate that C202A mutation diminishes the biochemical interaction between GSH and MaAKT1 channels induced by FSA treatment. Data are shown as mean ± SE (n = 3). Student’s t-test, ***p < 0.001 compared with the WT group. (F) Immunofluorescence showing that C202A mutation reduces the biochemical interaction between GSH and MaAKT1 channels induced by FSA treatment (n = 5). Bar = 20 μm.
Fig 8.
Cys202 mutation alleviates Foc TR4 and FSA-induced yellowing symptoms.
(A) Representative phenotypes, (B-C) root length, and (D) K+ fluxes measured from epidermal root cells of WT, atakt1 mutant, atakt1/MaAKT1, and atakt1/MaAKT1-C202A lines grown on MS medium with or without 10 μM FSA in Arabidopsis. (E) Disease symptoms and (F) DI distribution of WT, atakt1 mutant, akt1/MaAKT1, and akt1/MaAKT1-C202A lines infected with Fo5176 at 7 dpi in Arabidopsis. Data in (B), (C), and (D) are shown as means ± SE (n = 10). Student’s t-test, **p < 0.01, ****p < 0.0001.
Fig 9.
Working model of Foc TR4-induced suppression of MaAKT1-mediated K+ uptake.
The secondary metabolite FSA secreted by Foc TR4 induces oxidative stress, leading to ROS accumulation in banana root cells through hijacking RBohD. This disrupts redox homeostasis, promoting the conversion of GSH to GSSG and inhibiting MaAKT1 channels via S-glutathionylation at Cys202 residue. Reduced MaAKT1 channel activity impairs K+ uptake and reduces K+ content in roots, ultimately causing yellowing symptoms.