Inhibitory Properties of Cysteine Protease Pro-Peptides from Barley Confer Resistance to Spider Mite Feeding

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.

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…Investigation of the dionain-1 pro-domain binding mode and inhibitory potential is of particular interest for future investigation. Because pro-domain peptides have been shown to confer potent and selective inhibition of homologous C1 proteases across species (71,72), such knowledge could be applied in the specific targeting of proteolytic processes. This may be relevant in the inhibition of prey proteases released during the Venus flytrap digestion process or in the field of cancer biology and beyond, where homologous C1 proteases play pivotal roles in tissue remodeling, and selective inhibitors are of great interest to pharmaceutical and therapeutic research.…”

Drought-stressed tomato plants trigger bottom-up effects on key mite pests

“…On one hand, a positive correlation has been reported between mite fecundity and the amino acid and sugar content on plants (Tulisalo, 1971;Dabrowski and Bielak, 1978;Wermelinger et al, 1991), supporting the "Plant stress hypothesis". On the other hand, drought induces the accumulation of defense compounds like IPs, PPO and POD in tomato (English-Loeb et al, 1997;Inbar et al, 2001) that are detrimental for mites (Santamaria et al, 2012(Santamaria et al, , 2015b, which is consistent with the "Plant Vigor Hypothesis". The only study on tomato was performed with A. lycopersici, which increased its population and the damage it causes in the plant when irrigation was reduced (Gispert et al, 1989).…”

“…Thus, tomato serine PIs may probably be targeting other physiological processes in mites, such as the regulation of growth and development (Santamaria et al, 2012). Indeed, both cysteine and serine PIs can be harmful when ingested by T. urticae (Carrillo et al, 2011;Santamaria et al, 2012Santamaria et al, , 2015b. Interestingly, we have found that when feeding on tomato, cysteine and aspartyl protease and α-amylase activities were higher in T. urticae mites of the TA strain, whereas aminopeptidase activity was higher in the TNA strain.…”

“…The serine (PI-I, WIPI-II, JIP-21) aspartyl (CDI) and cysteine (CysPI) PI genes are induced by tetranychid mites in tomato but not by A. lycopersici (Li, et al, 2002;Kant et al, 2014;Glas, 2014;Glas et al, 2014;Alba et al 2015;Martel et al, 2015). Plant cysteine PIs are harmful for T. urticae (Santamaria et al, 2012(Santamaria et al, , 2015b been studied using some of these markers genes Alba et al, 2015). The JA response is in general fast, varying between 6h and 4 days depending on the severity of mite infestation Alba et al, 2015).…”

“…The serine (PI-I, WIPI-II, JIP-21) aspartyl (CDI) and cysteine (CysPI) PI genes are induced by tetranychid mites in tomato but not by A. lycopersici (Li, et al, 2002;Kant et al, 2014;Glas, 2014;Glas et al, 2014;Alba et al 2015;Martel et al, 2015). Plant cysteine PIs are harmful for T. urticae (Santamaria et al, 2012(Santamaria et al, , 2015b, as they inhibit some of the main mite gut proteolytic enzymes (cathepsins B-, L-, and D-and legumainlike proteases) (Carrillo et al, 2011;Santamaria et al, 2015a). Plant serine PIs are also harmful to T. urticae (Santamaria et al, 2012), but they may target other physiological processes, since serine proteases (trypsin and chymotrypsin) do not appear to be directly involved in the hydrolysis of dietary proteins in this species (Carrillo et al, 2011;Santamaria et al, 2015a).…”