Partitioning the structural features that underlie expansin-like and elicitor activities of cerato-platanin

Cerato-platanin family (CPF) proteins are produced by fungi and elicit defences when applied to plants, behaving as PAMPs/MAMPs. CPF proteins share structural similarity to plant and bacterial expansins, and have been demonstrated, in some cases, to possess expansin-like loosening activity on cellulose. This is the case of cerato-platanin (CP), the founder of the CPF, which shows both eliciting and cellulose-loosening activities, raising the question as to whether the expansin-like activity may be responsible for defence activation. To pinpoint structural and thermodynamic features underlying eliciting and expansin-like activity of CP, we carried out site-directed mutagenesis targeting separately net charge (N84D mutation), conformational stability (V63A mutation), or conserved position previously shown to affect expansin-like activity in CP (D77A mutation), and characterized wild-type protein and its variants. Removing or adding negative charges on the protein surface led to reducing or increasing, respectively, the expansin-like activity. The activity was instead not affected by mutations affecting protein fold and stability. In contrast, all the mutants showed reduced capacity to elicit defences in plants. We conclude that the expansin-like activity of CP depends on net charge and ability to (weakly) bind cellulose, whereas the eliciting activity on plants does not depend on the cellulose-loosening activity.