A tandem K unitz protease inhibitor ( KPI 106)–serine carboxypeptidase ( SCP 1) controls mycorrhiza establishment and arbuscule development in M edicago truncatula

Summary Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a K unitz protease inhibitor ( KPI 106) and a serine carboxypeptidase ( SCP 1) controls arbuscular mycorrhiza development in the root cortex of M edicago truncatula . Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza‐specific. Furthermore, the interaction between KPI 106 and SCP 1 analysed using the yeast two‐hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P 1 residue in KPI 106 ( L ys173) that fits into the catalytic pocket of SCP 1, suggesting that KPI 106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the L ys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference ( RNA i) inactivation of the serine carboxypeptidase SCP 1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI 106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP 1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI 106 also at the apoplast would modulate the spatial and/or temporal activity of SCP 1 by competing with the protease substrate.