Recombinant β-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth

Abstract

In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a β-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of β-1,3-1,4-glucanases and presented high protein identity with β-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with β-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches' broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao β-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a β-1,3-1,4-glucanase, in particular against M. perniciosa.

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

“…With regard to the hosts, 10 Theobroma cacao proteins of biotechnological interest have been characterized in vitro: four cystatins [ 117 ], one TcPR-10 [ 118 ], one β-1,3-1,4-Glucanase (TcGlu2) [ 119 ], one PR-4b [ 120 ], and one osmotin [ 121 ]. All of them have been shown to have a fungicidal effect on M. perniciosa .…”

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

“…EGases hydrolyse b-1,3-glucosidic bonds in b-1,3glucans, which are structural components of the cell walls of pathogenic fungi and oomycetes (Adams, 2004;Wessels, 1994). EGases are thus believed to directly hydrolyse pathogen cell walls, causing the lysis of pathogen cells (Britto et al, 2013). Accordingly, enzymes belonging to Class I have been reported to inhibit the growth of fungi in vitro (Arlorio et al, 1992;Ebrahim et al, 2011).…”

Rhodotorula mucilaginosa—alternative sources of natural carotenoids, lipids, and enzymes for industrial use

“…Moreover, R . mucilaginosa also could be developed as an industrial enzyme producer, especially in lipase and endo-β-1, 4-glucanases ( Britto et al., 2013 , Chennupati et al., 2009 ). Contingent on the cultivated environments, it has the capability to produce varied kinds of enzymes whose emergence may be industrially useful ( Zhang and Cui, 2012 ; Wu et al., 2013 ; Lario et al., 2015 ).…”