The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Trichoderma longibrachiatum Triggers a NAADP-Mediated Calcium Signalling Pathway in Lotus japonicus

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca(2+) increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca(2+)-dependence of these gene expression was demonstrated by using the extracellular Ca(2+) chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca(2+) elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca(2+)-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca(2+) release in a signalling pathway triggered by a biotic stimulus.