Caio Fernando Ramalho de Oliveira

Insecticidal Activity of Plant Lectins and Potential Application in Crop Protection

Lectins constitute a complex group of proteins found in different organisms. These proteins constitute an important field for research, as their structural diversity and affinity for several carbohydrates makes them suitable for numerous biological applications. This review addresses the classification and insecticidal activities of plant lectins, providing an overview of the applicability of these proteins in crop protection. The likely target sites in insect tissues, the mode of action of these proteins, as well as the use of lectins as biotechnological tools for pest control are also described. The use of initial bioassays employing artificial diets has led to the most recent advances in this field, such as plant breeding and the construction of fusion proteins, using lectins for targeting the delivery of toxins and to potentiate expected insecticide effects. Based on the data presented, we emphasize the contribution that plant lectins may make as tools for the development of integrated insect pest control strategies.

Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus

Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests.

Adaptive mechanisms of insect pests against plant protease inhibitors and future prospects related to crop protection: a review.

The overwhelming demand for food requires the application of technology on field. An important issue that limits the productivity of crops is related to insect attacks. Hence, several studies have evaluated the application of different compounds to reduce the field losses, especially insecticide compounds from plant sources. Among them, plant protease inhibitors (PIs) have been studied in both basic and applied researches, displaying positive results in control of some insects. However, certain species are able to bypass the insecticide effects exerted by PIs. In this review, we disclosed the adaptive mechanisms showed by lepidopteran and coleopteran insects, the most expressive insect orders related to crop predation. The structural aspects involved in adaptation mechanisms are presented as well as the newest alternatives for pest control. The application of biotechnological tools in crop protection will be mandatory in agriculture, and it will be up to researchers to find the best candidates for effective control in long-term.

Purification of a Kunitz-type inhibitor from Acacia polyphyllaDC seeds: characterization and insecticidal properties against Anagasta kuehniella Zeller (Lepidoptera: Pyralidae).

Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ∼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ∼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.

Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control.

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg(64) for the Glu(64) residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively.

A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is active against Spodoptera frugiperda larvae

A novel Kunitz-type inhibitor from Platypodium elegans seeds (PeTI) was purified and characterized. The mass spectrometry analyses of PeTI indicated an intact mass of 19 701 Da and a partial sequence homologous to Kunitz inhibitors. PeTI was purified by ion exchange and affinity chromatographies. A complex with a 1:1 ratio was obtained only for bovine trypsin, showing a Ki = 0.16 nM. Stability studies showed that PeTI was stable over a wide range of temperature (37-80 °C) and pH (2-10). The inhibitory activity of PeTI was affected by dithiothreitol (DTT). Bioassays of PeTI on Spodoptera frugiperda showed negative effects on larval development and weight gain, besides extending the insect life cycle. The activities of digestive enzymes, trypsin and chymotrypsin, were reduced by feeding larvae with 0.2% PeTI in an artificial diet. In summary, we describe a novel Kunitz inhibitor with promising biotechnological potential for pest control.

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI-induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI-induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.

Characterization of a Kunitz trypsin inhibitor from Enterolobium timbouva with activity against Candida species

Scientific advances have not been sufficient to accompany the growing resistance to antimicrobial medicines. High mortality rates due to opportunistic infections have threated human health. The development of new drugs, such as those obtained from plant sources, is a world priority. Herein, we report the purification of a trypsin inhibitor from Enterolobium timbouva seeds (EtTI) with regard to its homology, physico-chemical and inhibitory properties. Furthermore, we evaluated its activity against Candida strains, opportunistic pathogens regularly found in hospital infections. EtTI belongs to the Kunitz family and inhibits two trypsin molecules simultaneously; a feature shared among double-headed Kunitz inhibitors. A high inhibitory activity against trypsin was determined (Ki = 0.5 nM), and refractory to digestion by pepsin. EtTI was candidicidal against C. albicans, C. buinensis and C. tropicalis, triggering disturbances on integrity of the plasma membrane and morphological alterations, presumably mediated via apoptosis. The presence of two reactive sites is an unusual feature detected in EtTI. Numerous diseases and pathologies involve changes in peptidase activities, encouraging studies with multifunctional inhibitors. Accordingly, the further exploration of EtTI could provide new insights into the Kunitz inhibitors and their applications in disease control.

EMPREGO DE INIBIDORES DE PROTEASE VEGETAIS COMO FERRAMENTA BIOTECNOLÓGICA ALTERNATIVA NO CONTROLE DE PRAGAS

Estatisticas dao-nos conta que em2050 apopulacao humana sera de 9,1 bilhoes de habitantes. Nestas circunstâncias, recursos basicos como agua e alimentos tornar-se-ao escassos. Em especial, o fornecimento de alimento para a populacao em rapida expansao sera um desafio para a humanidade. Ha muito tempo o homem busca maneiras de combater as perdas agricolas. A introducao dos inseticidas quimicos nas lavouras foi muito importante, no entanto, seus efeitos toxicos sao indesejados. Por isso, a busca por agentes inseticidas que nao apresentem efeitos indesejaveis sobre a saude e ao meio ambiente tornou-se necessaria. Dentre as possibilidades existentes, a biotecnologia de plantas aparece como uma importante ferramenta para o controle de pragas, auxiliando no oferecimento de alimento com uma menor carga de produtos toxicos. Os inibidores de proteases constituem uma interessante classe de proteinas vegetais bioinseticidas com potencial biotecnologico. Durante decadas seu uso no combate a pragas agricolas vem sendo estudado e plantas transformadas expressando diferentes inibidores apresentaram resultados satisfatorios, mostrando perspectivas animadoras de sua aplicacao como ferramenta biotecnologica para a protecao dos cultivares. Nesta revisao apresentamos algumas informacoes sobre a biologia destas moleculas vegetais e sua historia, relatos de trabalhos onde sua atividade inseticida foi estudada em plantas modificadas geneticamente e ainda o futuro desta tecnologia, que podera envolver a combinacao de diferentes proteinas inseticidas e ate mesmo a fusao de proteinas para a obtencao de plantas mais eficientes.