Characterization of a cDNA encoding cystatin with antifungal activity from Siam tulip Curcuma alismatifolia

Abstract

Cystatins are cysteine protease inhibitors involved in defence mechanisms against pests and pathogens. Here, the cystatin CaCPI gene was isolated from a cDNA library of the Siam tulip (Curcuma alismatifolia cv. Chiang Mai Pink was then transformed into Escherichia coli strain BL21-Star to produce a recombinant CaCPI protein. After induction with 1 mM IPTG, the cell lysate of E. coli-carrying pDEST17-CaCPI generated a CaCPI protein about 12 kDa in size as measured using SDS-PAGE. Pre-incubation of the 5-30 µM CaCPI protein samples with 5 µM papain is known to decrease papain activity. Antifungal activities of the purified recombinant CaCPI protein against phytopathogenic fungi were tested. The CaCPI protein could suppress mycelium growth of Fusarium oxysporum, Colletotrichum capsici, and Pyricularia grisea phytopathogenic fungi.

In vitroeffect of recombinant amaranth cystatin (AhCPI) on spore germination, mycelial growth, stress response and cellular integrity ofAspergillus nigerandAspergillus parasiticus

“…However, A. niger was more susceptible, since it required a lesser AhCPI concentration to inhibit spore germination compared with A. parasiticus . Accordingly, it has been suggested that the cystatin inhibitory effect is determined by the fungal species (Porruan et al 2013); this hypothesis is in agreement with this study and others (Joshi et al 1998; Pernas et al 2000; Martínez et al 2003; Valdés-Rodríguez et al 2010). These results may be explained in terms of cell wall organization and composition, which are extremely variable and depend on many factors such as environmental changes, developmental stage, fungal species, etc.…”

“…The recombinant barley and chestnut cystatin inhibited the growth of Botrytis cinerea, Colletotrichum graminicola, Septoria nodorum and Plectosphaerella cucumerina ; interestingly, it did not affect Trichoderma viride (Pernas et al 2000; Martínez et al 2003). In the case of cystatin from siam tulip, the protein abolished mycelial growth of F. oxysporum, Colletotrichum capsici and Pyricularia grisea at different levels; 5 μM inhibited almost 100% of P. grisea whereas, F. oxysporum and C. capsici required almost 10 μM to reach the same level of inhibition (Porruan et al 2013). The differences in sensivity to cystatin between species could be given by developmental stage and/or cell wall composition (see below).…”

“…They have been related with defence mechanisms against insects and plant pathogens as well as plant response to heat, cold and saline stresses (Pernas et al 2000; Carrillo et al 2011; Valdés-Rodríguez et al 2015). It is well documented that phytocystatins present antifungal activity against a wide range of pathogenic fungi such as Botritys cinerea, Plectosphaerella cucumerina, Colletotrichum graminicola, Colletotrichum capsisi, Pyricularia grisea, Fusarium oxysporum, Septoria nodorum, Phytophthora nicotianae, Sclerotiana sclerotiorum, Sclerotium rolfsii, Alternaria brassicae, Glomerella cingulata, Pythium aphanidermatum, Rhizoctonia solani, Trichoderma reesei, Aspergillus sydowii, Helminthosporium sesamum (Pernas et al 1999; Sugawara et al 2002; Martínez et al 2003; Yang & Yeh 2005; Abraham et al 2006; Porruan et al 2013; Cheng et al 2014). However, the mechanism by which phytocystatins inhibit fungal growth is not yet known.…”

Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.)

“…For Hv-CPI1, FaCPI-1 and KCPI, 50% growth inhibition of B. cinerea occurred at concentrations of 1.5 μM, 1.9 μM and 2.7 μM, respectively [ 10 , 42 , 43 ], which are close to that of BnCPI (~1.81 μM). For F. oxysporum , 50% growth inhibition of HvCPI-1, AhCPI and CaCPI occurred at concentrations of 2.14 μM, 2.28 μM and 13 μM, respectively [ 10 , 18 , 44 ]. The 50% growth inhibition of reBnCPI on F. oxysporum was about 7.23 μM, which is higher than that reported for other phytocystatins.…”