Tenuazonic acid from Stemphylium loti inhibits the plant plasma membrane H⁺-ATPase by a mechanism involving the C-terminal regulatory domain

• Pathogenic fungi often target the plant plasma membrane (PM) H⁺-ATPase during infection. To identify pathogenic compounds targeting plant H⁺-ATPases, we screened extracts from 10 Stemphylium species for their effect on H⁺-ATPase activity. • We identified Stemphylium loti extracts as potential H⁺-ATPase inhibitors, and through chemical separation and analysis, tenuazonic acid (TeA) as a potent H⁺-ATPase inhibitor. By assaying ATP hydrolysis and H⁺ pumping, we confirmed TeA as a H⁺-ATPase inhibitor both in vitro and in vivo. To visualize in planta inhibition of the H⁺-ATPase, we treated pH-sensing Arabidopsis thaliana seedlings with TeA and quantified apoplastic alkalization. • TeA affected both ATPase hydrolysis and H⁺ pumping, supporting a direct effect on the H⁺-ATPase. We demonstrated apoplastic alkalization of A. thaliana seedlings after short-term TeA treatment, indicating that TeA effectively inhibits plant PM H⁺-ATPase in planta. TeA-induced inhibition was highly dependent on the regulatory C-terminal domain of the plant H⁺-ATPase. • Stemphylium loti is a phytopathogenic fungus. Inhibiting the plant PM H⁺-ATPase results in membrane potential depolarization and eventually necrosis. The corresponding fungal H⁺-ATPase, PMA1, is less affected by TeA when comparing native preparations. Fungi are thus able to target an essential plant enzyme without causing self-toxicity.