TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens

Abstract

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10–7 mol L−1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70–80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL−1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

State of the Art of the Molecular Biology of the Interaction between Cocoa and Witches’ Broom Disease: A Systematic Review

“…TcCYSPR04 [ 122 ] was also characterized in vitro but showed no significant fungicidal effect. Another protein from Theobroma cacao , the Kunitz-type trypsin inhibitor, was tested in vitro for its biotechnological potential and efficiency, but only as a potential larvicide against Helicoverpa armigera [ 123 ].…”

TcSERPIN, an inhibitor that interacts with cocoa defense proteins and has biotechnological potential against human pathogens

“…The in vitro inhibitory activity of the serpin was determined according to the optimum conditions for the activity of proteases (papain and trypsin), which were determined previously ( Pirovani et al., 2010 ; do Amaral et al., 2022 ), with adaptations. Against Carica papaya papain, the analyses were carried out in 50 mM phosphate buffer, pH 6.0, with 10 mM β-mercaptoethanol and 2 mM EDTA.…”

“…In plants, many of these inhibitors act as storage proteins ( Ostergaard et al., 2000 ; Grosse-Holz and van der Hoorn, 2016 ), control important endogenous proteases during seed germination ( Grosse-Holz and van der Hoorn, 2016 ; Tanner et al., 2019 ) and modulate plant tolerance to various stresses ( Benchabane et al., 2010 ; Koh et al., 2016 ; Martinez et al., 2016 ; Lema Asqui et al., 2018 ). In addition, many of these inhibitors have biotechnological potential against phytopathogens or agricultural pests, making them potential targets for agronomic studies ( Alvarez-Alfageme et al., 2011 ; do Amaral et al., 2022 ).…”

Genetic manipulation of anti-nutritional factors in major crops for a sustainable diet in future

“…Although there were some alterations in inhibitors, there were no changes in in the high-molecular-weight glutenin subunits or in yield ( Kalunke et al., 2020 ). However, trypsin inhibitors such as TcTI from cocoa provide significant defense against Helicoverpa ( do Amaral et al., 2022 ), and trypsin inhibitors that hinder digestion were also recently reported to be effective biopesticides ( Rodríguez-Sifuentes et al., 2020 ). Advanced gene editing techniques targeting two seed-specific KTI genes, namely KTI1 and KTI3 , resulting in small deletions and insertions in soybean open reading frames, offer an alternate strategy, by focusing on reducing trypsin inhibitors only in seeds for consumption, will be helpful in the future ( Wang et al., 2022 ).…”