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.

Ana Camila Oliveira Freitas,Cristiane Ferreira Souza,Alex Alan Furtado De Almeida,Wanius Garcia,Carlos Priminho Pirovani,Paulo Sérgio Monzani,Marcio Gilberto Cardoso Costa,Rogerio Margis

doi:10.1371/journal.pone.0121519

Ana Camila Oliveira Freitas, Cristiane Ferreira Souza + Show 6 more

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https://doi.org/10.1371/journal.pone.0121519

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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.

Highlights

The fermented seeds of cocoa (Theobroma cacao L), known as beans, are considered a commodity, since they serve as raw material for the chocolate industry
We evaluated the effects of heat treatments on the recombinant TcCYS1, TcCYS2, TcCYS3, and TcCYS4 cystatins from T. cacao, in relation to the cysteine protease inhibition activity and the formation of dimers
We analyzed the effect of heat treatments and pH variation on the secondary structure and activity of these proteins named TcCYS1, TcCYS2, TcCYS3, and TcCYS4 [13]

Summary

Introduction

The fermented seeds of cocoa (Theobroma cacao L), known as beans, are considered a commodity, since they serve as raw material for the chocolate industry. The witche’s broom disease (WBD) caused by the fungus Moniliophthora perniciosa is an important disease in cocoa producing areas of Central and South America [1,2]. The biotrophic or parasitic phase is characterized by the presence of monocariotic mycelium in the intercellular space, causing the loss of apical dominance, hyperplasia, hypertrophy, and proliferation of axillary branches called green brooms [4]. In the necrotrophic or saprophytic phase, the hyphae are slender, dicariotic, and present clamp connections between the septa. At this stage, the plant’s infected tissue is necrotic and dead, forming dry brooms [4,5,6,7,8]

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