Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed

Cysteine-proteases and cystatins from barley: molecular and functional characterization in housekeeping and defense processes

Plant cysteine-proteases (CysProt) represent a well-characterized type of proteolytic enzymes that fulfill tightly regulated physiological functions (senescence and seed germination among others) and defense roles. This article is focused on the group of papain-proteases C1A (family C1, clan CA) and their inhibitors, phytocystatins (PhyCys). In particular, the protease–inhibitor interaction and their mutual participation in specific pathways throughout the plant's life are reviewed. C1A CysProt and PhyCys have been molecularly characterized, and comparative sequence analyses have identified consensus functional motifs. A correlation can be established between the number of identified CysProt and PhyCys in angiosperms. Thus, evolutionary forces may have determined a control role of cystatins on both endogenous and pest-exogenous proteases in these species. Tagging the proteases and inhibitors with fluorescence proteins revealed common patterns of subcellular localization in the endoplasmic reticulum–Golgi network in transiently transformed onion epidermal cells. Further in vivo interactions were demonstrated by bimolecular fluorescent complementation, suggesting their participation in the same physiological processes.