The Activity of TcCYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa, Caused by the Fungus Moniliophthora perniciosa

Abstract

The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.

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

“…Previous observations have shown that serpins from barley and wheat grains, for example, are apparently inactivated at more acidic pH levels ( Roberts et al., 2003 ). Changes in acidity and temperature are efficient strategies for inactivating plant defense proteins during Theobroma cacao-Moniliophthora perniciosa interaction ( Freitas et al., 2015 ).…”

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

“…Recently, it was shown that variations in pH and temperature can affect the oligomerization of TcCYS4 modifying the inhibitory activity this cystatin against PLPCs engaged in PCD, and affect symptoms of witches’ broom disease of cocoa, caused by the fungus M . perniciosa [ 54 ]. Cystatin levels also decrease in mature tissues, while the main difference concerns the protein levels between infected and uninfected tissues during the transition from green broom to dry broom, which corresponds to the transition from biotrophic to necrotrophic mycelium of M .…”

Protease inhibitors from Theobroma cacao impair SARS-CoV-2 replication in vitro

“…Cysteine-protease inhibitors are candidates to treat SARS-CoV and MERS-CoV [ 12 ]. Protease inhibitors from Theobroma cacao have been characterized and described as papain like protease inhibitors, called TcCYS3 and TcCYS4, two thermally stable cystatins [ 13 ] that can also inhibit the growth of Moniliophtera perniciosa [ 14 ].…”