Isoforms of cysteine peptidases and osmotins instead of chitinases and peptidase inhibitors drive antifungal activity but occur as distinct isoforms in latex and callus tissues of Calotropis procera.

Peptidases and osmotins are the primary determinants of antifungal activity, and their expression is tightly linked to tissue specialization in Calotropis procera plants. This study investigated whether callus cultures of two Apocynaceae species, Calotropis procera and Cryptostegia grandiflora, reproduce the antifungal defense system typically found in their latex. Protein fractions obtained from latex and callus tissues were evaluated for antifungal activity against Fusarium solani, F. oxysporum, Colletotrichum lindemuthianum, and C. gloeosporioides, as well as the presence of pathogenesis-related (PR) proteins was investigated. Callus proteins from C. procera (CPCp) exhibited antifungal activity, inhibiting both mycelial growth and spore germination of all tested fungi. In contrast, callus proteins from C. grandiflora (CPCg) showed no detectable antifungal activity. Mechanistic assays demonstrated that CPCp induced plasma membrane permeabilization and the accumulation of reactive oxygen species in fungal spores. Biochemical analyses further revealed that antifungal activity was associated with the presence of peptidases and osmotins but not of peptidase inhibitors and chitinases. Immunological recognition and gene expression analyses indicated that the peptidases and osmotins expressed in callus tissues differ from those present in the latex of C. procera, suggesting the occurrence of distinct protein isoforms. Collectively, these findings demonstrate that peptidases and osmotins are key determinants of antifungal activity in latex plants and reveal that their expression is closely linked to tissue specialization, highlighting the differential regulation of defense protein isoforms between latex and other plant tissues.