Inhibition of Plant-Pathogenic Fungi by the Barley Cystatin Hv-CPI (Gene Icy) Is Not Associated with Its Cysteine-Proteinase Inhibitory Properties

Abstract

The recombinant barley cystatin Hv-CPI inhibited the growth of three phytopathogenic fungi (Botrytis cinerea, Colletotrichum graminicola, and Plectosphaerella cucumerina) and the saprotrophic fungus Trichoderma viride. Several mutants of barley cystatin were generated by polymerase chain reaction approaches and both their antifungal and their cysteine-proteinase inhibitory properties investigated. Point mutants R38-->G, Q63-->L, and Q63-->P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C68-->G was more active for papain and cathepsin B than the wild type. These results indicate that in addition to the consensus cystatin-reactive site, Q63-V64-V65-A66-G67, the A37-R38-F39-A40-V41 region, common to all cereal cystatins, and the C68 residue are important for barley cystatin activity. On the other hand, the K92-->P mutant is inactive as a fungicide, but still retains measurable inhibitory activity for papain and cathepsin B. Against B. cinerea, the antifungal effect of Hv-CPI and of its derived mutants does not always correlate with their activities as proteinase inhibitors, because the Q63-->P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K92-->P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteine-proteinase inhibitory activity. Moreover, these results are corroborated by the absence of inhibition of intra- and extramycelia-proteinase activities by barley cystatin and by other well-known inhibitors of cysteine-proteinase activity in the fungal zymograms of B. cinerea.

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 phytocystatin isolated from taro corms ( Colocasia esculenta ) was able to inhibit mycelial growth and cause the lysis of sclerotia in Sclerotium rofsii (Yang & Yeh 2005). 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).…”

“…In opposition, evidence from Hv-CPI showed that the antifungal effect of this cystatin and its derived mutants does not correlate with their activities as proteinase inhibitors. Recombinant Hv-CPIs affected in their activities as proteinase inhibitors due to point mutations (R38 → G, Q63→L, and Q63→P) conserved its antifungal properties (Martínez et al 2003; Cheng et al 2014). In the same way, no correlation was found between the inhibition of papain activity and the antifungal effect of different segments of the tarocystatins (Wang et al 2008).…”

“…It was proved that AhCPI was able to inhibit the growth of some phytopathogenic fungi such as Fusarium oxysporum, Sclerotium cepivorum and Rhyzoctonia solani ; however, this effect was not related to its ability to inhibit the activity of mycelial proteases (Valdés-Rodríguez et al 2010). In agreement, the antifungal effect of barley cystatin (Hv-CPI) and its derived mutants does not correlate with their activities as proteinase inhibitors (Martínez et al 2003). All of these results are supported by evidences presented by Wang et al, based on the inhibitory kinetics of different segments of tarocystatins on papain activity.…”

“…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.…”