Beatriz Godoy Roncalli ¹, Lucas Carvalho de Freitas ¹, Roberto Afonso da Silva ², Tercilio Calsa Junior ¹

Opuntia stricta, or prickly pear, is a cactus species adapted to the Brazilian semiarid region, tolerant to water deficit and higher temperatures, but susceptible to biotic stresses such as infestation by the armored scale insect pest (Diapsis echinocacti), which may cause severe physiological damage to the plant. In response, prickly pear plants activate defense pathways including the synthesis of protease inhibitors (PIs), key proteins in defense against herbivory and other biotic stresses. Therefore, this study aimed to identify potential PIs in O. stricta subjected to infestation by armored scale insects. Proteins from the aqueous extract of  Opuntia stricta clone IPA200016 cladodes infested by D. echinocacti were obtained through precipitation at 0–30% and 30–60% (w/v) ammonium sulfate saturation levels. Subsequently, the proteins from these fractions were subjected to enzymatic digestion with trypsin and analyzed by nUPLC-MS/MS mass spectrometry in the Q-ToF Synapt XS system (Waters, Inc.). The resulting spectra were processed, and proteins were identified with the help of the Progenesis QI v4.7 software (Nonlinear Dynamics). The analysis of the conserved domains of the proteins was performed with CD-Search tool, using the Pfam conserved domains database. Among the proteins identified in the aqueous extract of O. stricta, seven types of PIs were found, which may be directly associated with infestation by D. echinocacti. Four proteins displayed conserved domains compatible with the beta-trefoil_STI superfamily, commonly associated with trypsin inhibitors and other serine proteases, suggesting their potential role in defense against sucking pest insect biotic stresses. Two proteins were related to the CY superfamily, characteristic of cystatins, known for their role as inhibitors of cysteine proteases. One of these proteins also showed similarity with domains of the PP1 superfamily, associated with phosphatases, which may indicate additional regulatory functions and multifunctionality of these inhibitors in the context of stress responses. Thus, the PIs identified in this study may be helpful for understanding the response of the prickly pear to D. echinocacti infestation, and also for strategies towards the development of management or improvement actions of resistance to biotic stresses.

Agradecimentos: I am grateful to my advisor, lab colleagues, and collaborators for all their support. Thank you very much to all of you.