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Transgenic sugarcane expressing V-ATPase subunit E dsRNA affects growth and survival of Sphenophorus levis. Plants being sessile organisms are constantly confronted with several biotic and abiotic stresses. Sugarcane (Saccharum spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to Sphenophorus levis (sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with V-ATPase E dsRNA in S. levis larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the V-ATPase E gene of S. levis in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.

Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes.

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.

Huanglongbing (HLB) is a devastating citrus disease associated with Candidatus Liberibacter asiaticus (CLas) and is transmitted by the psyllid Diaphorina citri Kuwayama. Diaphorina citri belongs to Hemiptera order, which has cysteine peptidases as the most abundant proteolytic enzymes present in digestive tract. As cysteine peptidases are involved in different insect development processes, this class of enzymes has acquired biotechnological importance. In this context, we identified a cathepsin L-like (DCcathL1) from the Diaphorina citri transcriptome database and expressed the enzyme in E. coli. Quantitative real-time RT-PCR was conducted to determine DCcathL1 gene expression in different parts and developmental phases of the insect. We observed that DCcathL1 expression in the gut was 2.59 and 2.87-fold higher than in the head and carcass, respectively. Furthermore, DCcathL1 expression was greater in eggs than in nymphs and adults, suggesting a putative role of the enzyme in the embryonic development. In addition, enzymatic inhibitory activity using four recombinant Citrus cystatins were performed. Among them, CsinCPI-2 was the strongest DCcathL1 inhibitor with a Ki value of 0.005 nM. Our results may contribute in the development of strategies for D. citri control, such as silencing the DCcathL1 gene and the use of transgenic plants that overexpress peptidase inhibitors.

Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

Saccharomyces cerevisiae is the most important microorganism used in the ethanol fermentation process. The PE-2 strain of this yeast is widely used to produce alcohol in Brazil due to its high fermentation capacity. The aim of the present study was to develop an expression system for recombinant proteins using the industrial PE-2 strain of S. cerevisiae during the alcoholic fermentation process. The protein chosen as a model for this system was CaneCPI-1, a cysteine peptidase inhibitor. A plasmid containing the CaneCPI-1 gene was constructed and yeast cells were transformed with the pYADE4_CaneCPI-1 construct. To evaluate the effect on fermentation ability, the transformed strain was used in the fermentation process with cell recycling. During the nine-hour fermentative cycles the transformed strain did not have its viability and fermentation ability affected. In the last cycle, when the fermentation lasted longer, the protein was expressed probably at the expense of ethanol once the sugars were exhausted. The recombinant protein was expressed in yeast cells, purified and submitted to assays of activity that demonstrated its functionality. Thus, the industrial PE-2 strain of S. cerevisiae can be used as a viable system for protein expression and to produce alcohol simultaneously. The findings of the present study demonstrate the possibility of producing recombinant proteins with biotechnological applications during the ethanol fermentation process.

Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (K m = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (K m = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K(m)) of 47 μM(-1)∙s(-1). The purified ((HIS))CPXAC displayed maximal catalytic activity at pH 5.5 and 30°C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K(i) values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.

Plant legumains, also termed vacuolar processing enzymes (VPEs), are cysteine peptidases that play key roles in plant development, senescence, programmed cell death and defense against pathogens. Despite the increasing number of reports on plant cysteine peptidases, including VPEs, the characterization of sugarcane VPEs and their inhibition by endogenous cystatins have not yet been described. This is the first report of the biochemical characterization of a sugarcane cysteine peptidase. In this work, a recombinant sugarcane legumain was expressed in Pichia pastoris and characterized. Kinetic studies of the recombinant CaneLEG revealed that this enzyme has the main characteristics of VPEs, such as self-activation and activity under acidic pH. CaneLEG activity was strongly inhibited when incubated with sugarcane cystatin 3 (CaneCPI-3). Quantitative analysis of CaneLEG and CaneCPI-3 gene expression indicated a tissue-specific expression pattern for both genes throughout sugarcane growth, with the strong accumulation of CaneLEG transcripts throughout the internode development. Furthermore, the CaneLEG and CaneCPI-3 genes exhibited up-regulation in plantlets treated with abscisic acid (ABA). These results suggest that CaneCPI-3 may be a potential endogenous inhibitor of CaneLEG and these genes may be involved in plant stress response mediated by ABA. Also, the expression analysis provides clues for the putative involvement of CaneLEG and CaneCPI-3 in sugarcane development and phytohormone response.

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37°C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K(m)=37.53 mMS(-1)), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K(i)=0.196 nM), indicating that this protease is a potential target for pest control.

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).