Potencial biotecnológico da toxina da soja (SBTX) como uma ferramenta contra fungos

Abstract

Soybean (Glycine max) is a species of great economic value for Brazil given the multiplicity of use of its seeds in animal feed and industry. Although Brazil is the second largest producer of seeds, the losses in productivity in the field are still considerable, especially those caused by pathogenic fungi, despite of the existence of alternatives to chemical control of these species and resistant genotypes. Thus, the search for natural mechanisms of resistance is an environmentally safe practice needed to control pests and pathogens and to improve the productivity. The present study aimed to evaluate the biotechnological potential of SBTX as a tool to control pathogenic fungi of agronomic importance, and to investigate the mechanism of action by which this protein exerts its activity. SBTX (44 kDa) was able to interfere with the growth of hyphae of the fungus Cercospora kikuchii at concentration of 50 pgP/mL. Nevertheless, this protein did not inhibit spore germination of Colletotrichum gloeosporioides, Phomopsis sp., and C. kikuchii even at a concentration of 200 pgP/mL. Since C. kikuchii is a fungus that attacks soybean leaves, the presence of SBTX in these organs was evaluated. In leaves of young plants were found 35,08 ± 0,57 pg of SBTX/mgP, whereas in mature leaves the content observed was 18,60 ± 2,89 pg of SBTX/mgP. SBTX was induced 24 hours after mechanical injury of leaves (from 35,08 ± 0,57 pg of SBTX/mgP to 209 ± 2.38 pg of SBTX/mgP), functioning as a defense protein. In view of the biotechnological potential of this protein in controlling pathogenic fungi, the present study also aimed to elucidate the mechanism of action of SBTX, using yeast as model. SBTX (200 pgP/mL) inhibited the growth of Candida albicans, but not the yeasts Pichia membranifaciens and Candida parapsilosis. However, morphological changes characterized as pseudohyphae were observed in P. membranifaciens and C. parapsilosis, promoted by SBTX (400 pgP/mL). The aggregation of cells was about ten times higher in P. membranifaciens and C. parapsilosis after incubation with SBTX compared to control. Moreover, SBTX caused membrane permeabilization in these cells guided by SYTOX Green and inhibited by about 27% the acidification stimulated by glucose in Saccharomyces cerevisiae cells. Cells of P. membranifaciens SBTX treated (400 mg/ mL) presented cell wall disruption, condensation and shrinkage of cytoplasm, in addition to increased vacuolation and loss of normal structure and cytoplasm content. Assessing the pattern of gene expression in cells of C. alb/cans was found in the ability of SBTX to induce the expression of genes involved in various cellular processes, including transport of molecules, metabolism of carbohydrates and amino acids, cycle and cellular respiration, as well as genes involved in responses to drugs and stress. Among the induced genes, must be reported to TUPI, which encodes a transcription factor capable of suppressing the formation of pseudohyphae, justifying the absence of such structures in C. albicans, even under stress conditions imposed by SBTX. The mechanism of inhibition of SBTX in C. albicans proposed here is that the toxin may have crossed the cell wall, activates the glucose sensor Hgt4 present in the cell membrane and compromisied, its assimilation, stimulating C. albicans to activate other metabolic pathways such as gluconeogenesis, beta-oxidation and Leloir pathway, and also interfered in the levels of other essential components to the cell, such as histones and RNA and induced the formation of reactive oxygen species.