Mitochondrial Signs and Subcellular Imaging Provide Insight into the Antifungal Mechanism of Carabrone against Gaeumannomyces graminis var. tritici

Abstract

Carabrone, a botanical bicyclic sesquiterpenic lactone, has broad-spectrum antifungal activity and is particularly efficient against the devastating phytopathogen Gaeumannomyces graminis var. tritici (Ggt). The antifungal mechanism of carabrone against Ggt, however, remains unclear. The main objective of this study was to investigate the subcellular localization of carabrone in Ggt to gain a better understanding of its mechanism of action. When Ggt was exposed to carabrone (EC value of 28.45 μg/mL) for 7 days, a decline in mitochondrial concentration together with some obvious alternations in mitochondrial structure, including hazy outlines, medullary transitions, excess accumulation of unclear settlings, and vacuolar degeneration, were observed, indicating that carbrone may act on the mitochondria directly. A fluorescent conjugate (TTY) was thus designed and synthesized as a surrogate of carabrone that possessed comparable antifungal activity against Ggt (EC of 33.68 μg/mL). Additionally, a polyclonal antibody specific to carabrone and with a high titer (256 000) was also prepared by immunizing mice. Subsequently, two imaging techniques, the use of the fluorescent conjugate (FC) and immunofluorescence (IF), were applied to determine the subcellular localization of carabrone. Both FC and IF fluorescent signals demonstrated its mitochondrial localization with a Pearson's coefficient of 0.83 for FC and 0.86 for IF. These results imply that carabrone exerts its antifungal activity against Ggt by interfering with mitochondrial function.

A functional analysis of mitochondrial respiratory chain cytochrome bc1 complex in Gaeumannomyces tritici by RNA silencing as a possible target of carabrone

“…In our previous study, we reported that carabrone exerts significant antifungal activity against G. tritici (Wang et al, 2017b(Wang et al, , 2017c. Using transmission electron microscopy, carabrone was found to have a significant effect on biological oxidation and caused mitochondrial vacuolation in G. tritici.…”

“…Immunogen (T-BSA) was synthesized according to the method described by Wang et al (2017b) Female BALB/c mice (6 weeks old), purchased from the Xi'an Jiaotong University School of Medicine (Shaanxi, China), were immunized with immunogen (T-BSA). Immunization of the animals and titres of the antisera were monitored by indirect ELISA, as described by Wang et al (2017b). The monoclonal antibody was prepared according to the method described by Wang et al (2017a) with slight modifications.…”

“…Using transmission electron microscopy, carabrone was found to have a significant effect on biological oxidation and caused mitochondrial vacuolation in G. tritici. Subsequently, we identified the mitochondria as the subcellular location of carabrone by confocal microscopic detection of a fluorescently labelled molecules (Wang et al, 2017b).…”

“…Carabrone displays antibacterial and antitumour activities, and is associated with low toxicity and few negative effects on environmental health (Feng et al ., 2010; Wang et al ., 2014). In our previous study, we reported that carabrone exerts significant antifungal activity against G. tritici (Wang et al ., 2017b, 2017c). Using transmission electron microscopy, carabrone was found to have a significant effect on biological oxidation and caused mitochondrial vacuolation in G. tritici .…”

“…Using transmission electron microscopy, carabrone was found to have a significant effect on biological oxidation and caused mitochondrial vacuolation in G. tritici . Subsequently, we identified the mitochondria as the subcellular location of carabrone by confocal microscopic detection of a fluorescently labelled molecules (Wang et al ., 2017b). We further reported that the mitochondrial respiratory chain complex III in G. tritici is highly sensitive to carabrone and is a potential target of carabrone (Wang et al ., 2017c).…”