A real or perceived reduction in intracellular glutamine levels restores appressorium formation to Δ<i>asd4</i> mutant strains.

<p>(A) Asd4 physically interacts with the <i>GLN1</i> promoter <i>in vivo</i> following growth on GMM with 10 mM NH₄⁺ as the sole nitrogen source. ChIP was performed using Anti-GFP. The eluted <i>GLN1</i> DNA signal, normalized against the input control, was significantly (<i>Student’s t-test</i> p ≤ 0.05) enriched in Δ<i>asd4 ASD4GFP</i> ChIP samples compared to WT controls lacking Asd4^GFP. (B) Steady-state intracellular concentrations of glutamine were quantified using LC-MS/MS analysis of amino acid extracts from dried fungal mycelia of the indicated strains following growth on GMM + 10 mM NH₄⁺ media. Values are the average of three independent biological replicates. (C) Appressorial development of WT, <i>Δasd4</i> and <i>Δasd4 Δgln1</i> strains on artificial hydrophobic surfaces (coverslips) at 24 hpi. Scale bar is 10 μm. (D) Appressorial formation rates of WT, <i>Δasd4</i> and <i>Δasd4 Δgln1</i> mutant strains on artificial hydrophobic surfaces (coverslips). (E) Treatment of Δ<i>asd4</i> spores with the glutamine synthetase inhibitor 10 mM L-methionine sulphoximine (MSX) partially remediated appressorium formation. (F) <i>Δasd4</i> mutant strains formed appressoria at 24 hpi on artificial hydrophobic surfaces following treatment with 55 nM rapamycin (Rap). (G) Steady-state intracellular concentrations of glutamine were quantified following growth of the indicated strains on GMM + 10 mM NH₄⁺ media with and without 55 nM rapamycin. Values are the average of three independent biological replicates. (H) Treating spore suspensions with 55 nM rapamycin induced appressorium formation by WT and Δ<i>asd4</i> mutant strains on non-inductive hydrophilic surfaces. NT is untreated. (A-B, D-H) Error bars are standard deviation. Bars with different letters are significantly different (<i>Student’s t-test</i> p ≤ 0.05). (D-F, H) Values are the average of the number of appressoria formed at 24 hpi from 50 spores per coverslip, repeated in triplicate.