Abstract
C1A plant cysteine proteases are synthesized as pre-pro-enzymes that need to be processed to become active by the pro-peptide claves off from its cognate enzyme. These pro-sequences play multifunctional roles including the capacity to specifically inhibit their own as well as other C1A protease activities from diverse origin. In this study, it is analysed the potential role of C1A pro-regions from barley as regulators of cysteine proteases in target phytophagous arthropods (coleopteran and acari). The in vitro inhibitory action of these pro-sequences, purified as recombinant proteins, is demonstrated. Moreover, transgenic Arabidopsis plants expressing different fragments of HvPap-1 barley gene containing the pro-peptide sequence were generated and the acaricide function was confirmed by bioassays conducted with the two-spotted spider mite Tetranychus urticae. Feeding trials resulted in a significant reduction of leaf damage in the transgenic lines expressing the pro-peptide in comparison to non-transformed control and strongly correlated with an increase in mite mortality. Additionally, the analysis of the expression levels of a selection of potential mite targets (proteases and protease inhibitors) revealed a mite strategy to counteract the inhibitory activity produced by the C1A barley pro-prodomain. These findings demonstrate that pro-peptides can control mite pests and could be applied as defence proteins in biotechnological systems.
Highlights
Among 20–30,000 genes encoded by a plant genome, almost one thousand correspond to proteases, and more than one hundred belong to the 15 known families of cysteine-proteases (CysProt) [1]
Individual C1A protease members are involved in a variety of proteolytic and physiological processes in plants such as senescence, abscission, programmed cell death, fruit ripening, pollen development, and the mobilization of proteins accumulated in seeds and tubers [6,7,8,9]
The inhibitory capability of eight recombinant pro-peptides derived from different barley C1A CysProt (HvPap-1pro, HvPap-4pro, HvPap-6pro, HvPap-10pro, HvPap-12pro, HvPap-16pro, HvPap-17pro and HvPap-19pro) was in vitro tested against crude protein extracts of three phytophagous coleopteran species (Leptinotarsa decemlineata, Diabrotica virgifera and Tenebrio molitor) and three phytophagous acari species (T. urticae, T. evansi and Brevipalpus chilensis)
Summary
Among 20–30,000 genes encoded by a plant genome, almost one thousand correspond to proteases, and more than one hundred belong to the 15 known families of cysteine-proteases (CysProt) [1]. Individual C1A protease members are involved in a variety of proteolytic and physiological processes in plants such as senescence, abscission, programmed cell death, fruit ripening, pollen development, and the mobilization of proteins accumulated in seeds and tubers [6,7,8,9]. Their implication in local and systemic defense responses against pathogens and pests has been published [10,11,12,13]. The relatively acidic pH of these compartments provides the optimal conditions for protease processing and activation by removing of the N-terminal pro-peptide
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