The antifungal activity of a wheat germin-like protein that inhibits trypsin involves the inhibition of extracellular fungal proteinases

MARCHETTI, M.F.; MANSILLA, A.Y.; CONDE, R.D.; MENDIETA, J.R.

Rosario, Santa Fe

Congreso; IX Congreso de Microbiologia General; 2013

Some serine proteinases inhibitors (SPI) from plants may display additional associated biological activities. Recently, it has beenreported that some SPI may control fungal growth participating in plant defense mechanism. However, the mode of action ofthese SPI is poorly understood. In our laboratory, a SPI has been purified from intercellular fluid of wheat leave and it shareshomology with the Germin Like-Proteins family (GLPIs) so it was named Germin Like Protein Inhibitor (GLPI). In addition to itsinhibitor activity, GLPI has at least two more activities linked to responses to different types of stress: superoxide dismutase(SOD) and adenosine glucose pyrophosphatase/phosphodiesterase (AGPPase), so GLPI is considered a multifunctionalprotein.Certain species of Fusarium spp. infect wheat spikes causing Fusarium head blight (FHB), a devastating disease of wheatspread worldwide that affects our country. A part from wheat spikes, both flowers and grains are affected by this fungus.Humidity and warm temperature during the grain filling period favors the fungal development reducing the crop yield.The aim of this study was to determine whether GLPI has antifungal activity against Fusarium solani trying to dilucidate theunderlying mechanism. Firstly, we analyzed the effect of GLPI on F. solani spores germination. In vitro quantitative assaysshowed that the growth inhibition produced by GLPI is dose dependent, displaying an estimated IC50 value (concentrationrequired to produce a 50% growth inhibition) of approximately 0.720 mg/ml. To investigate whether GLPI has a lethal effect, twoexperimental approaches were performed. First, F. solani spores were incubated overnight in the presence of differentconcentrations of GLPI. Then, the incubation mix was plated on potato dextrose agar plates. The fungal viability was calculatedcounting the CFU. As a second approach, the effect of GLPI on fungal plasma membrane was assayed based on the uptake ofthe fluorogenic dye SYTOSX Green (it only penetrates cells that have damaged plasma membranes and fluoresces uponbinding to DNA). The results obtained suggest that GLPI is not acting as a fungicide, but instead, has a fungistatic effect.Besides, GLPI does not disturb the fungal plasma membrane. These observations prompted us to investigate if the antifungalproperty of GLPI is a consequence of its activity as a serine proteinase inhibitor acting on a fungal proteinase. Hence, the totalazocaseinolytic activity of extracellular F. solani proteinases in the presence of GLPI was evaluated. The results showed thatGLPI inhibited the total azocaseinolytic activity by a 50%. All together, the results obtained in this study suggest that GLPI exertsa fungistatic effect against F. solani by the inhibition of an extracellular proteinase/s required for its normal germination.