br Conflict of interest br Acknowledgments We thank

Conflict of interest
Acknowledgments We thank Jianru Zuo (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for providing fbr11-1 seeds. We are grateful to Carol MacKintosh and the DSTT at the University of Dundee (UK) for anti-RD21 antibodies. This work was supported by the Centre National de la Recherche Scientifique, the Université de Toulouse, the French Laboratory of Excellence project “TULIP” (ANR-10-LABX-41; ANR-11-IDEX-0002-02) and “SPS” (ANR-10-LABX-0040-SPS) and the ERC Consolidator grant 616449 ‘GreenProteases’. K. Y. K. was supported by the IFE-STDF call 4/2016-2017 fellowship.
Malaria is an important human infection, resulting in 429 thousand deaths in 2015 . Resistance to drugs appears early in the parasite population and inhibits the control of this disease. Approximately 100 proteases were identified in the genome , as proteolysis is tightly associated with intraerythrocytic cycle events, such as invasion and egress of erythrocytes, degradation of host proteins and inactivation of host immune defense mediators . Three cysteine proteases from , falcipain-1, falcipain-2 and falcipain-3, have been well-characterized , . Localized in the food vacuole, falcipains-2 and falcipain-3 are essential for hemoglobin digestion, which is crucial for amino ceramide kinase acquisition , and osmotic stability of the parasite . Cystatins are competitive inhibitors of cysteine proteases and represent a major class of proteases inhibitors, the cystatin superfamily . Phytocystatins or plant cystatins are considered a distinct family and can be found in a variety of superior plants. Several biological functions have been attributed to phytocystatins, including regulation of endogenous proteinases and defensive roles against insects and fungi , . Sugarcane cystatins were first described by Reis et al. , who identified 25 cystatin-like sequences in the Sugarcane Expressed Tags Project (SUCEST) database . Four sugarcane cystatins (CaneCPI-1, CaneCPI-2, CaneCPI-3 and CaneCPI-4) were recombinantly expressed and c , , . Among them, CaneCPI-4 showed the highest inhibitory activity against human cathepsins B and L, attenuating the invasive activity of breast cancer cells . In addition, CaneCPI-4 showed high inhibitory potential against a recombinant cathepsin L from (), a sugarcane pest . Here, we evaluated the ability of CaneCPI-4 to inhibit development in cultures by reducing the cysteine protease proteolytic activity on hemoglobin. Isolated parasites or infected erythrocytes were incubated with recombinant CaneCPI-4 (MW=13kDa) labeled with the fluorescent probe FITC (isocyanate of fluorescein). The protein uptake in the cytoplasm of isolated parasites (A) and inside -infected erythrocytes (B) was assessed by fluorescence microscopy. Fluorescent-labeled CaneCPI-4 (CaneCPI-4-FITC) was selectively internalized by the parasites, while non-infected erythrocytes did not show any labeling (B). The cystatin internalization was also confirmed by CaneCPI-4 antibody recognition by confocal imunofluorescence (C). These data corroborate previous findings that can uptake proteins and peptides from extracellular medium , . Furthermore, did not cleave the cystatin as shown by Western blotting analysis (D), suggesting that the whole protein is resistant to proteolysis in iRBC until reaches the parasite cytoplasm. To test this hypothesis, isolated parasites in suspension (10cells/mL) were incubated with CaneCPI-4, and the intracellular proteolytic activity was determined using the fluorogenic substrate -Phe-Arg-MCA (Z=carbobenzoxy; MCA=7-amino-4-methylcoumarin) (). This approach is advantageous because the inhibitor was assayed against the proteases in their natural environment, the parasite cytoplasm. We observed that at least 1μM of CaneCPI-4 inhibited approximately 70% of the intracellular protease activity, while 10, 25 or 50μM of the inhibitor nearly abolished the proteolytic activity (A). The inhibition is specific to the cystatin, as the addition of an anti-CaneCPI-4 antibody completely blocked the inhibition (B).