Abstract
Magnaporthe oryzae is an agricultural mold that causes disease in rice, resulting in devastating crop losses. Since rice is a world-wide staple food crop, infection by M. oryzae poses a serious global food security threat. Fungicides, including azole antifungals, are used to prevent and combat M. oryzae plant infections. The target of azoles is CYP51, an enzyme localized on the endoplasmic reticulum (ER) and required for fungal ergosterol biosynthesis. However, many basic drug-pathogen interactions, such as how the azole gets past the fungal cell wall and plasma membrane, and is transported to the ER, are not understood. In addition, reduced intracellular accumulation of antifungals has consistently been observed as a drug resistance mechanism in many fungal species. Studying the basic biology of drug-pathogen interactions may elucidate uncharacterized mechanisms of drug resistance and susceptibility in M. oryzae and potentially other related fungal pathogens. We characterized intracellular accumulation of azole drugs in M. oryzae using a radioactively labeled fluconazole uptake assay to gain insight on whether azoles enter the cell by passive diffusion, active transport, or facilitated diffusion. We show that azole accumulation is not ATP-dependent, nor does it rely on a pH-dependent process. Instead there is evidence for azole drug uptake in M. oryzae by a facilitated diffusion mechanism. The uptake system is specific for azole or azole-like compounds and can be modulated depending on cell phase and growth media. In addition, we found that co-treatment of M. oryzae with ‘repurposed’ clorgyline and radio-labeled fluconazole prevented energy-dependent efflux of fluconazole, resulting in an increased intracellular concentration of fluconazole in the fungal cell.
Highlights
The fungal plant pathogen Magnaporthe oryzae is considered to be one of the most dangerous and destructive agricultural molds
For a final efflux-related analysis, we evaluated the effects of combination treatment of M. oryzae with 3H-FLC and clorgyline, a drug once used as an antidepressant that has recently been shown to have efflux inhibitor properties (Youdim, 1975; Holmes et al, 2012)
E-test strips were used to establish M. oryzae susceptibility to common medical antifungals belonging to different classes of drugs and determine if a study using medical azoles was relevant in M. oryzae
Summary
The fungal plant pathogen Magnaporthe oryzae is considered to be one of the most dangerous and destructive agricultural molds. Fungicides, including agricultural azoles such as propiconazole, prothioconazole, tebuconazole, and prochloraz, are used to combat rice blast (Talbot, 2003; Wilson and Talbot, 2009; Edwards and Godley, 2010; Parker et al, 2011, 2013; Kaur et al, 2012; Lehoczki-Krsjak et al, 2013, 2015). Fungicides are utilized as both seed treatment to prevent infection of the seedlings after germination, and as foliar sprays on mature leaves to prevent infection and spread during the growing season (Couch et al, 2005; Edwards and Godley, 2010; Parker et al, 2011, 2013; Kaur et al, 2012; Lehoczki-Krsjak et al, 2013, 2015)
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