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

Abstract

Summary 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.

A critical review on natural compounds interacting with the plant plasma membrane H+‐ATPase and their potential as biologicals in agriculture

“…TeA also overcame activation of the PM H + ‐ATPase by FC in a competition assay, suggesting that the C‐terminal regulatory domain is targeted by TeA. Further analysis indicated that TeA targets part of the C‐terminal regulatory domain of the PM H + ‐ATPase somewhere between the last 77 and 40 amino acid residues (Figure 5B), making the PM H + ‐ATPase able to bind fewer 14‐3‐3 proteins (Bjork et al, 2020).…”

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid

“…TeA has several molecular targets in the cells of various organisms. In plant cells, it inhibits the proton pump of the plasma membrane and photosynthesis [ 80 , 81 ], being one of the pathogenicity factors of phytopathogenic fungi that produce it [ 82 ]. Cell-free assays demonstrated TeA to be an antioxidant and a promising inhibitor of acetylcholinesterase and β-amyloid aggregation [ 83 , 84 ].…”

Recent Advances in Alternaria Phytotoxins: A Review of Their Occurrence, Structure, Bioactivity, and Biosynthesis

“…In the last two decades, an increasing number of articles have reported its phytotoxicity. TeA showed an inhibitory activity against 4-hydroxyphenylpyruvate dioxygenase (HPPD) with an IC 50 of 18 µM [237] and plant plasma membrane (PM) H + -ATPase [238]. TeA was also able to inhibit the elongation of seedling roots and shoots [239][240][241], and resulted in a significant increase in multi-nucleolus of Vicia faba root tip cells at 400 µg•mL −1 [242].…”