The fungal plant pathogen Magnaporthe oryzae is considered to be one of the most dangerous and destructive agricultural molds. Named after the host it infects, M. oryzae causes disease in rice (Oryza sativa) called rice blast, which can infect all parts of the rice plant causing lesions on leaves, stems, seeds, and even roots (Martin-Urdiroz et al., 2016; Yan and Talbot, 2016). Hundreds of millions of people world-wide depend on rice as a staple food crop making rice the most important grain with regard to human diet and nutrition (Harrison, 2002; Thornton and Wills, 2015). Consequently, rice blast poses a serious global food security threat. So great is the potential risk for crop failure or severely reduced crop yield due to rice blast, that M. oryzae has been ranked among the most significant plant pathogens (Talbot, 2003; Thornton and Wills, 2015).

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 also 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).

The target of azole antifungals is the fungal Cyp51, Lanosterol 14α-demethylase. This enzyme is a member of the cytochrome P450 superfamily of enzymes required for ergosterol biosynthesis in fungi.

Drugs that have intracellular targets, such as Cyp51 located on the endoplasmic reticulum (ER), must cross both the fungal cell wall and the plasma membrane and then be transported to its ER destination. It is not clear how azoles are able to do this. It has generally been supposed that azoles enter the cell by passive diffusion, until recent analysis of fluconazole (FLC) uptake in C. albicans and A. fumigatus by our lab argue against a passive diffusion mechanism (Mansfield et al., 2010; Esquivel et al., 2015). Identifying drug uptake mechanisms in fungi opens many new avenues for investigation in basic fungal biology, pathogenesis, and new drug design. Insights into new fungal-specific drug targets, or finding treatment strategies using combinations of existing drugs, is of great importance to control the spread of resistant fungi.

Importantly to our research, M. oryzae shares many characteristics associated with other notable cereal pathogens (Talbot, 2003; Wilson and Talbot, 2009). Advances in understanding the genes and mechanisms that govern the resistance and susceptibility in this species, will help advance our understanding of other related fungal diseases.

In this study, we extended our previously reported radioactively labeled fluconazole accumulation assay to measure cellular accumulation of azole drugs in M. oryzae and determine if azoles enter the cells by passive diffusion, active transport, or a facilitated diffusion mechanism. Consistent with our import experiments on the filamentous human fungal pathogen A. fumigatus, our import experiments with M. oryzae have shown that azole drugs do not accumulate in the fungal cell solely by passive diffusion or ATP-dependent active transport. Instead there is evidence for azole import by a facilitated diffusion mechanism such as a protein carrier or channel.

In addition to uptake analysis, we used ³H-FLC to measure glucose-stimulated and glucose-independent azole efflux over time from M. oryzae. The M. oryzae strain used in this study is the first plant pathogenic fungus to be fully sequenced allowing a direct examination of its genome (Dean et al., 2005). Compared to yeast species such as Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Candida glabrata, and Candida krusei, the filamentous fungus M. oryzae contains an unusually high number of genes encoding predicted membrane transporters. This includes at least 50 ATP-Binding Cassette (ABC) and at least 250 Major Facilitator Superfamily (MFS) transporters (Coleman and Mylonakis, 2009; Kim et al., 2013). The majority of these transporter genes are still uncharacterized to date (Kovalchuk and Driessen, 2010; Kim et al., 2013).

Our ³H-FLC efflux data showed evidence for a glucose-stimulated azole efflux mechanism in M. oryzae, which would be consistent with an ABC superfamily transporter activity.

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). These results demonstrate the potential usefulness of combination drug therapies and the idea of repurposing archived compounds for alternative uses as the threat of resistance to current fungal treatments emerges.

There is a continual emergence of fungicide-resistant pathogens in clinical and agricultural isolates (Fleming et al., 2002). Understanding even the most basic cellular processes and epidemiology of these isolates is imperative to prevent their spread, determine treatment and prevention strategies, optimize future drug design, and to predict future evolution of resistance (Ashkani et al., 2011).

Many fungi with intrinsic resistance to antifungal agents already exist in our environment (36). We illustrated this with E-test susceptibility testing of M. oryzae, which displayed FLC and CFG resistance (Figure 1). Most filamentous fungal species and molds are intrinsically resistant to FLC since they possess multiple target CYP51 paralogs: two in M. oryzae, A. fumigatus, and A. nidulans, and three in A. flavus and species of Fusarium (Lepesheva and Waterman, 2007). This CYP51 redundancy allows for slight changes to occur in the active site of one or all Cyp51 copies that affects the binding affinity to azoles (Hawkins et al., 2014). The structure of FLC in particular allows multiple Cyp51 active site binding conformations that are weak or transitory so that there is incomplete inhibition of the Cyp51 target enzyme compared to other azoles that have stronger Cyp51 binding affinity (Fleming et al., 2002; Mellado et al., 2005; Cools et al., 2010; Warrilow et al., 2010; Parker et al., 2011; Yan et al., 2011; Hawkins et al., 2014).

Regardless, the E-tests confirmed that even azole drugs used to treat human fungal pathogens are taken up by the plant pathogen M. oryzae as evidence by growth inhibition seen with the medical azoles ITC, KTC, POS, and VRC. Overall, the susceptibility and resistance patterns illustrated by the E-tests suggest a common mechanism of action of azoles on all fungal species, including the requirement for entry into the cell, passing through the cell wall and plasma membrane. This may be the first time reporting MICs of medical antifungals on a distinctly plant pathogen.

In this work, we have begun to analyze potential molecular mechanisms of azole drug resistance in the filamentous plant pathogen M. oryzae by characterizing azole import into the fungal cell under a variety of environmental conditions, as well as azole efflux from the cell, using radioactively labeled FLC. Azoles must enter the fungal cell in order to inhibit the intracellular Cyp51 target enzyme. Therefore reduced or modified drug import may help to explain why some pathogenic fungi are more resistant to azoles than others. Our assay can be used to compare drug import in agricultural, medical and other pathogenic fungi.

Our experiments thus far have demonstrated that azole entry into the fungal cell is not solely by a passive diffusion mechanism. There may be some baseline level of azole passive diffusion into the cell but our evidence suggests azoles import into M. oryzae is more substantially through a plasma membrane-localized protein channel or carrier. Likewise, there may be a certain amount of azole import due to uncharacterized ATP-dependent importers that was masked by the high activity of ATP-dependent efflux transporters. Drug uptake was observed in de-energized cells (Figure 3A). However, media that contained glucose showed reduced final drug accumulation levels, presumably due to activation of glucose-dependent efflux pumps.

Our data strongly support the idea of azole entry into the cell by facilitated diffusion via a membrane protein channel or carrier that recognizes a specific moiety found in azole drugs. This finding is in agreement with studies on the human pathogenic fungi C. albicans, C. neoformans, and A. fumigatus as well as the model yeast S. cerevisiae (Mansfield et al., 2010; Esquivel et al., 2015).

Import of azoles did not require a proton gradient as no change was observed in uptake over a range of buffered pHs (Figure 3C). There was a trend toward alkaline sensitivity for drug uptake as seen by a decrease in ³H-FLC uptake in samples at pH 7 media. However, a deficiency in cell growth was observed in M. oryzae cells at pH 7, so import at this pH may be affected by other cellular factors directly or indirectly related to pH and proton gradients.

We did find significant differences in drug accumulation in M. oryzae depending on the growth media used, as shown in Figure 3B. Environmental adaptations that prevent or enhance azole uptake is an important aspect for drug resistance and treatment analysis. Based on these import results and a comparison of the components of these three medias, it is difficult to identify a single factor that would cause such a dramatic difference in uptake between CSM compared to YAD and YPD medias. The cell adaptations to the different media that affect FLC uptake is probably a complex mixture of differences in protein synthesis, lipid storage, transcriptional activity, and other metabolic activities that may alter the cell membrane composition. The altered azole uptake between the samples argues against passive diffusion entry into the cell, in which case one would expect only minimal reduction or increase in drug accumulation between the samples.

A comparison of drug import in exponential vs. post-exponentially growing cells (Figure 3D) shows that fluconazole accumulation is cell phase dependent. Drug accumulation in the older, post-exponential or stationary growing cells is dramatically reduced compared to the exponentially growing cells. This is consistent with major differences in cell metabolic activity when comparing exponentially growing cells and stationary phase cells, including changes to transcription, protein translation, modifications, and secretion, membrane maintenance, and other vital cell processes.

Exponentially growing cells are considered more active and responsive, while stationary phase cells shift to a period of maintenance and conservation (Werner-Washburne et al., 1993). It has been shown for some drugs, that proliferating cells are more sensitive than those cells in a steady state, so an increase in intracellular drug concentrations in exponential phase cells compared to stationary cells would correspond with those observations (43). This could be responsible for the decrease in azole uptake in exponential phase cell growth. The differential uptake of azoles into exponential cell phase is evidence against passive diffusion in which there would be no cell phase dependency.

Strong evidence for a saturable protein carrier is illustrated by competitive inhibition of ³H-FLC uptake by other azoles (Figure 4). ³H-FLC import was significantly inhibited by simultaneous treatment with an excess of unlabeled azoles. However, other antifungal drugs did not compete for import into the cell and may even stimulate uptake of azoles. This indicates substrate specificity for moieties found in the azole structure. The structure of competitive inhibitors and drugs that did not compete for import are shown in Tables 2, 3, respectively.

It is important to understand how different compounds are taken into the cell for the purposes of dosing and increasing treatment effectiveness, and also for combination therapy considerations. A combination of drugs that have the same mode of action but different mechanisms of uptake, may have an effect on synergy of the drugs. Antagonism may also be a result of competition for import into the cell.

Taking the ³H-FLC assay in another direction, we used radioactively labeled FLC to measure azole release from azole-preloaded hyphal cells. Our evidence indicates that the efflux of azoles is stimulated by energy (Figure 5) potentially via ABC efflux transporters, which suggests there are distinct transporters for influx and efflux of azoles, as opposed to a single transporter which functions in both directions.

The ABC superfamily of efflux pumps are well characterized for their ability to carry a broad range of substrates including, but not limited to, antifungal drugs across biological membranes (Decottignies et al., 1994; Holland and Blight, 1999; Wilkens, 2015). Resistance to commonly used antifungals has frequently been shown to develop due to overexpression or increased activity of ABC transporters in human fungal pathogens (Cannon et al., 2009; Sanglard, 2016). More recently, ABC transporters are being recognized for their role in pathogenicity, virulence, stress tolerance, and drug resistance in M. oryzae and other plant pathogens (Urban et al., 1999; Sun et al., 2006; Gupta and Chattoo, 2008; Price et al., 2015).

Much of the efflux of ³H-FLC from M. oryzae in glucose-stimulated and glucose-independent conditions occurred before the 4 h time point and so earlier time points would be useful for an in-depth analysis of efflux kinetics in M. oryzae (Figure 5).

The addition of clorgyline, a compound in the LOPAC collection and potential inhibitor of energy-dependent efflux, showed reduced efflux of ³H-FLC from energized M. oryzae cells (Figure 6). The ability of a compound to increase antifungal drug accumulation in the cell or prevent the rapid efflux of a drug makes it an ideal candidate to be considered for a synergistic combination therapy. A more thorough examination of clorgyline as an efflux inhibitor is needed, but our and others’ preliminary data suggest that clorgyline, or similar compounds, could potentially be used as combination therapy in either agriculture or clinical practice. The notion of considering medically now-outdated drugs for use as agricultural treatments unlocks many new possibilities. It might be more practical to reconsider ‘failed’ human treatment strategies, whether due to pharmacologic properties of absorption, distribution, adherence, etc. in humans, for use in plant fungal pathogens. Repurposing compounds from the LOPAC collection would shave years off the drug development process as these compounds have previously been well characterized, synthesized, stabilized, and near optimized.

Design of the study: TW and BE. Experimental work: BE. Data analysis and interpretation: TW and BE. Writing the manuscript: BE. Final approval: TW and BE.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.